Insecticidal proteins derived from bacillus thuringiensis

ABSTRACT

The present invention relates to the field of plant pest control, particularly insect control. Provided are nucleotide sequences from  Bacillus thuringiensis  encoding insecticidal proteins. Further provided are methods and means for using said nucleotide sequence for controlling plant insect pests.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of application Ser. No. 11/892,796, filedAug. 27, 2007, which is a division of application Ser. No. 10/392,874filed Mar. 21, 2003, which claims priority under 35 USC 119 to U.S.Provisional Application No. 60/336,276, entitled Novel BacillusThuringiensis Insecticidal Proteins and filed on Mar. 22, 2002, and U.S.Provisional Application No. 60/423,999, entitled Novel BacillusThuringiensis Insecticidal Proteins, and filed on Nov. 6, 2002. Theentire contents of these priority applications are incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to the field of plant pest control,particularly insect control. Provided are new nucleic acid sequencesderived from Bacillus thuringiensis (Bt) strains, encoding insecticidalproteins expressed during vegetative growth stages. Particularly, DNAsequences encoding proteins designated as ISP3-1099E, ISP3-327D andISP3-2245J are provided, which are useful to protect plants from insectdamage. Further provided are plants and microorganisms comprising atleast one of the new nucleic acid molecules, as well as methods andmeans for using these nucleic acid sequences for reducing insect damageof plants.

BACKGROUND ART

Insect pests cause huge economic losses worldwide in crop production,and every year farmers face the threat of yield losses due to insectinfestation. Genetic engineering of insect resistance in agriculturalcrops has been an attractive approach to reduce costs associated withcrop-management and chemical control practices. The first generation ofinsect-resistant crops was introduced into the market in 1996, based onthe expression in plants of proteins isolated from the gram-positivesoil bacterium Bacillus thuringiensis (Bt). The insecticidal Bt Cryproteins are produced during the sporulation-stage of Bt strains and theproteins accumulate in large cytoplasmic crystals within the bacterium.When taken up by insects, a typical Lepidopteran-toxic Bt Cry protein issolubilized and processed in the insect midgut into an active form ofabout 60 to 65 kDa. The active protein exerts its toxic effect bybinding to the midgut epithelial cells, causing pore formation in thecell membrane, which leads to osmotic lysis of the cells (Gill et al.,1992).

A Bt strain may produce many different toxins. Since the isolation ofthe first insecticidal crystal protein-encoding gene from Bt in 1981(Schnepf and Whiteley, 1981), more than 100 Bt Cry toxin-encoding geneshave been cloned and insect pests have been effectively controlled byexpressing Bt-derived proteins in agricultural important crop species.However, the use of individual Bt proteins is often limited, as most Btproteins are active against only a relatively small number of thenumerous insect pests. Specificity of Bt Cry proteins is thought to bedetermined by factors such as the activation of the toxin in the insectgut (Haider et al. 1986) and its ability to bind specific receptors(Hofmann et al., 1988).

The risk that susceptible insect species may develop resistance againstBt Cry toxins is widely recognized. Consequently, active efforts havebeen made to identify novel insecticidal proteins. One strategy that hasbeen used was to screen Bacillus strains for the production ofinsecticidal proteins during vegetative growth stages, rather thanduring sporulation stages. Using this approach, a number of “vegetativeinsecticidal proteins” or “VIPs” have been identified.

Estruch et al. (1996), WO94/21795, WO96/10083, WO98/44137, U.S. Pat. No.5,877,012, U.S. Pat. No. 6,107,279, U.S. Pat. No. 6,137,033 and U.S.Pat. No. 6,291,156 describe the isolation of vip3A(a), vip3A(b) andvip3A(c) from supernatant fluids of Bt strains AB88, AB424 and AB51.

According to the authors, these genes encode proteins with insecticidalactivity towards a broad range of Lepidopteran insect pests.

WO98/18932 and WO99/57282 describe a number of nucleotide sequencesisolated from Bt strains. These sequences are referred to as mis (mis-1to mis-8), war and sup. According to the authors, the encoded proteinshave activity against Lepidopteran or Coleopteran pests.

WO00/09697 describes heat-labile, soluble MIS-type and WAR-type toxins,as well as smaller (1 to 10 kDa) toxins, obtainable from the supernatantof cultures of Bacillus laterosporus strains, which, according to theauthors, have activity against Western Corn Rootworm larvae.

WO98/00546 and U.S. Pat. No. 6,274,721 describe the isolation of Btstrains and Bt toxins, which, according to the authors, have activityagainst Lepidopteran pests.

WO99/33991 describes the isolation of Bt strains and Bt toxins, which,according to the authors, have activity against Lepidopteran pests.

Recently, Selvapandiyan et al. (2001) described the isolation of a geneencoding a protein designated as VIP-S. According to the authors theVIP-S protein showed toxicity against a number of Lepidopteran insectspecies.

Doss et al. (2002) describe the cloning of VIP3V from strain Btkurstaki.

WO02/078437 describes VIP3 toxins from Bt, such as VIP3A, VIP3B andVIP3A-B hybrid toxins.

Despite the isolation and characterization of a relatively large numberof different insecticidal proteins to date, there remains a need foridentification, isolation and characterization of new insecticidalproteins. The reasons for this are manifold. Firstly, due to thespecificity of insecticidal proteins towards particular groups of targetpests (host insect spectra), there is a need to clone genes encodingproteins with different spectra of activity, so that for different cropsand different geographic regions suitable proteins for combating insectpests are available. The specificity of Bt Cry proteins, for example, ismostly limited. Identification of toxins with specificity towardsdifferent target insects remains desirable. Second, after prolonged usein one geographic region, insects are known to have the capacity todevelop resistance towards chemical insecticides and microbial sprays(for example based on Bt spore-crystal mixtures), and are believed tohave the capacity to develop resistance towards plants expressinginsecticidal proteins. The development of resistance within insectpopulations could render existing insecticidal proteins ineffective,creating a need for novel genes and proteins. Third, for health andenvironmental reasons it is desirable to identify proteins with high,specific insecticidal potency and acute bioactivity towards targetinsect species.

The present invention provides, including the different embodimentsdescribed in the claims, novel nucleic acid sequences and amino acidsequences isolated from Bacillus thuringiensis strains. These nucleicand amino acid sequences are useful to protect plants from insectdamage, either by the expression of the nucleic acid sequences withinplants under the control of suitable promoters, or by externalapplication of the toxins to the plants. The toxins of the subjectinvention are distinct from previously-described pesticidal toxins.

SUMMARY OF THE INVENTION

The present invention provides insecticidal ISP3 proteins and nucleicacids encoding them. In particular, the present invention providesinsecticidal proteins ISP3-1099E (SEQ ID NO: 2), ISP3-327D (SEQ ID NO:4) and ISP3-2245J (SEQ ID NO: 6). The present invention also providesnucleic acids encoding those proteins, such as isp3-1099E (SEQ ID NO:1), isp3-327D (SEQ ID NO: 3) and isp3-2245J (SEQ ID NO: 5),respectively. The proteins of the present invention have insecticidalactivity against Lepidopteran insect pests. Particular insectssusceptible to the proteins of the present invention include Helicoverpazea, Helicoverpa armigera, Helicoverpa punctigera, Heliothis virescens,Ostrinia nubilalis, Spodoptera frugiperda, Agrotis ipsilon, Pectinophoragossypiella, Scirphophaga incertulas, Cnaphalocrocis medinalis, Sesamiainferens, Chilo partellus, Anticarsia gemmatalis, Plathypena scabra,Pseudoplusia includens, Spodoptera exigua, Spodoptera omithogalli,Epinotia aporema and Rachiplusia nu.

Another embodiment of the present invention provides insecticidalvariants and fragments of the ISP3 proteins, and of the nucleic acidsencoding them. Such variants include, for example, nucleic acidsequences that hybridize under stringent conditions to SEQ ID NO: 1, SEQID NO: 3 or SEQ ID NO: 5.

In one embodiment of the invention insecticidal proteins comprising atleast 91% sequence identity to SEQ ID NO: 2, at least 91% sequenceidentity to SEQ ID NO: 4, or at least 88% sequence identity to SEQ IDNO: 6 are provided.

In a further embodiment of the invention, isolated nucleic acidsequences comprising at least 93% sequence identity to SEQ ID NO: 1, atleast 94% sequence identity to SEQ ID NO: 3, or at least 97% sequenceidentity to SEQ ID NO: 5 are provided.

In yet a further embodiment nucleic acid sequences encoding the ISP3proteins of the invention are provided, whereby the nucleic acidsequence is a synthetic sequence which has been optimized for expressionin monocotyledonous or dicotyledonous plants or plant cells.

The present invention also provides chimeric genes comprising a promotersequence operably linked to a nucleic acid sequence encoding an ISP3protein, particularly ISP3-1099E, ISP3-327D or ISP3-2245J orinsecticidally active variants or fragments thereof. Also provided arevectors comprising nucleotide sequences encoding ISP3 proteins,particularly ISP3-1099E, ISP3-327D, or ISP3-2245J, or insecticidallyactive variants or fragments thereof.

The invention further provides host cells comprising chimeric genesencoding ISP3 proteins, particularly ISP3-1099E, ISP3-327D, orISP3-2245J, or insecticidally active fragments or variants thereof. Suchhost cells may be microorganisms or transgenic plant cells, planttissues, plant organs, plant seeds or whole plants. The transformedplant is any plant. In one embodiment, the transformed plant is a maizeor cotton plant. In an alternative embodiment, the transformed plant isa rice or soybean plant. Other suitable plants include, but are notlimited to, sorghum, wheat, barley, rye, sunflower, sugarcane, tobacco,Brassica species (such as oilseed rape, mustard, cabbage, broccoli,etc.), vegetable species (tomato, cauliflower, radish, spinach, pepper,onion, bean, pea, carrot, etc.), sugar beet, tree species (apple, pear,plum, conifers, deciduous trees etc.), potato, alfalfa, mango, papaya,or banana.

The present invention also provides methods of protecting a plantagainst insect damage, comprising contacting said plant with aninsecticidal ISP3 protein. The plant may be contacted with an ISP3protein by transforming the plant with a nucleotide sequence encoding anISP3 protein, or by applying an ISP3 protein externally to the plant.

In one embodiment of the present invention a Bt strain comprising anisp3 nucleic acid, particularly isp3-1099E, isp3-327D or isp3-2245J isprovided.

In a further embodiment, an insecticidal composition comprising aninsecticidally effective amount of ISP3 protein is provided. Whenapplied externally to a plant, the insecticidal composition increasesresistance to insect damage in treated plants, compared to untreatedcontrol plants, to which no such composition is applied.

In a further embodiment, the present invention provides a method ofevolving a nucleic acid sequence encoding an ISP3 protein is provided.This method comprises the steps of:

-   a) providing a population of nucleic acid sequences encoding the    amino acid sequences of SEQ ID NO: 2 and/or SEQ ID NO: 4 and/or SEQ    ID NO: 6, or variants or fragments of the amino acid sequences of    SEQ ID NO: 2 and/or SEQ ID NO: 4 and/or SEQ ID NO: 6, wherein said    variants or fragments have a sequence identity of at least 91% to    SEQ ID NO: 2 or 4 and at least 88% to SEQ ID NO: 6;-   b) shuffling said population of variants or fragments to form    recombinant nucleic acid molecules;-   c) selecting or screening for recombinant nucleic acid molecules    that encode proteins that have insecticidal activity; and    -   repeating steps (a) to (c) with the recombinant nucleic acid        molecules selected in step (c) until a recombinant nucleic acid        molecule encoding a protein having the desired insecticidal        property has been found.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present invention provides methods and means for reducing damage toplants caused by pests, particularly insect pests, such as lepidopteraninsect pests. The present invention further provides novel nucleic acidsequences and proteins that are distinct from previously describednucleic acid sequences and proteins. These nucleic acids and proteinscan be used for controlling insect pests, either by integration andexpression of at least one of these new nucleotide sequences in plantsor plant cells, or by external treatment of plants or plant parts withcompositions comprising the toxins encoded by these nucleic acidmolecules.

The present invention provides novel pesticidal toxins derived fromBacillus thuringiensis strains. The pesticidal toxins of the presentinvention include the proteins designated as ISP3-1099E protein;ISP3-327D protein; and ISP3-2245J protein.

In accordance with this invention, a “nucleic acid sequence” refers to aDNA or RNA molecule, in single- or double-stranded form, that encodesany of the ISP3 proteins of this invention. It is clear that the basesequence of an RNA molecule is identical to the base sequence of thecorresponding DNA molecule, with the difference that any T (thymine)residues in the DNA molecule are replaced by U (uracil) residues in theRNA molecule. The term “isolated nucleic acid sequence”, as used herein,is not limited to a nucleic acid sequence in isolation, but alsoencompasses a nucleic acid sequence that is no longer in the naturalenvironment where it was isolated from. Thus, an “isolated nucleic acidsequence” includes the nucleic acid sequence in another bacterial hostor in a plant nuclear genome.

In accordance with the present invention, the terms “protein” or“polypeptide” are used interchangeably to refer to a molecule consistingof a chain of amino acids, without reference to any specific mode ofaction, size, three-dimensional structure or origin. Hence, a fragmentor portion of an ISP3 protein of the invention is still referred toherein as a “protein”. The phrase “isolated protein”, as used herein, isnot limited to a protein in isolation, but also encompasses a proteinthat is no longer in its natural environment. The natural environment ofthe protein refers to the environment in which the protein could befound when the nucleotide sequence encoding it was expressed andtranslated in its natural environment, i.e. in the environment fromwhich the nucleotide sequence was isolated. For example, an isolatedprotein can be present in vitro, or in another bacterial host or in aplant cell, or it can be secreted from another bacterial host or from aplant cell.

In accordance with this invention, nucleic acid sequences, including DNAsequences, encoding new ISP3 proteins have been isolated andcharacterized. The new genes were designated isp3-1099E, isp3-327D andisp3-2245J and their encoded proteins ISP3-1099E, ISP3-327D andISP3-2245J, respectively.

In accordance with this invention, “ISP3-1099E protein” refers to anyprotein comprising the smallest fragment of the amino acid sequence ofSEQ ID NO: 2 that retains insecticidal activity (hereinafter referred toas the “smallest toxic fragment”). This includes hybrid or chimericproteins comprising the smallest toxic fragment. Also included in thisdefinition are variants of the amino acid sequence in SEQ ID NO: 2, suchas amino acid sequences essentially similar to SEQ ID NO: 2, having asequence identity of at least 91%, or at least 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% at the amino acid sequence level. In the context of thepresent invention, “sequence identity” may be determined using pairwisealignments using the GAP program of the Wisconsin package of GCG(Madison, Wis., USA, version 10.2). The GAP program is used with thefollowing parameters for the amino acid sequence comparisons: the‘blosum62’ scoring matrix, a ‘gap creation penalty’ (or ‘gap weight’) of8 and a ‘gap extension penalty’ (or ‘length weight’) of 2. Insecticidalproteins according to the present invention may have some, for example5-10, or less than 5 amino acids added, replaced or deleted withoutsignificantly changing, or without changing the insecticidal activity ofthe protein. Changes in the amino acid sequence that do not change theinsecticidal activity of the protein in a negative way are also includedin this definition.

In accordance with this invention “ISP3-327D protein” refers to anyprotein comprising the smallest fragment of the amino acid sequence ofSEQ ID NO: 4 that retains insecticidal activity (hereinafter referred toas “smallest toxic fragment”). This includes hybrid- or chimericproteins comprising the smallest toxic fragment. Also included in thisdefinition are variants of the amino acid sequence in SEQ ID NO: 4. Suchvariants may include essentially similar amino acid sequences, having asequence identity of at least 91%, or at least 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% at the amino acid sequence level, as determined usingpairwise alignments using the GAP program of the Wisconsin package ofGCG (Madison, Wis., USA, version 10.2). The GAP program is used with thefollowing parameters for the amino acid sequence comparisons: the‘blosum62’ scoring matrix, a ‘gap creation penalty’ (or ‘gap weight’) of8 and a ‘gap extension penalty’ (or ‘length weight’) of 2. Preferably,proteins having some, e.g. 5-10, or less than 5, amino acids added,replaced or deleted without significantly changing the insecticidalactivity of the protein, or at least without changing the insecticidalactivity of the protein in a negative way, are included in thisdefinition.

In accordance with this invention, “ISP3-2245J protein” refers to anyprotein comprising the smallest fragment of the amino acid sequence ofSEQ ID NO: 6 that retains insecticidal activity (hereinafter referred toas “smallest toxic fragment”). This includes hybrid or chimeric proteinscomprising the smallest toxic fragment. Also included in this definitionare variants of the amino acid sequence in SEQ ID NO: 6. Such variantsinclude amino acid sequences essentially similar to SEQ ID NO: 6, havinga sequence identity of at least 88%, or at least 89%, 90%, 91%, 92%,93%, 95%, 96%, 97%, 98% or 99% at the amino acid sequence level, asdetermined using pairwise alignments using the GAP program of theWisconsin package of GCG (Madison, Wis., USA, version 10.2). The GAPprogram is used with the following parameters for the amino acidsequence comparisons: the ‘blosum62’ scoring matrix, a ‘gap creationpenalty’ (or ‘gap weight’) of 8 and a ‘gap extension penalty’ (or‘length weight’) of 2. This definition includes proteins having some,e.g., 5-10, or less than 5, amino acids added, replaced or deletedwithout significantly changing the insecticidal activity of the protein.Sequence changes that do not change the insecticidal activity of theprotein in a negative way are also included in this definition.

As used herein, the term “comprising” is to be interpreted as specifyingthe presence of the stated features, integers, steps or components asreferred to, but does not preclude the presence or addition of one ormore features, integers, steps or components, or groups thereof. Thus,reference herein to DNA or protein “comprising the sequence or region X”refers to a DNA or protein including or containing at least the sequenceor region X, so that other nucleotide or amino acid sequences can beincluded at the 5′ (or N-terminal) and/or 3′ (or C-terminal) end. Forexample, a nucleotide sequence comprising DNA encoding a selectablemarker protein as disclosed in EP 0 193 259, may comprise the nucleotidesequence encoding a transit peptide, and/or a 5′ or 3′ leader sequence.

The “smallest toxic fragment” of an ISP3 protein of the invention, asused herein, refers to the smallest fragment or portion of an ISP3protein retaining insecticidal activity that can be obtained byenzymatic digestion of the full length ISP3 protein. “Smallest toxicfragment” also encompasses the smallest fragment or portion of an ISP3protein retaining insecticidal activity that can be obtained by makingnucleotide deletions in the DNA encoding an ISP3 protein. DNA encodingshorter toxic ISP3 fragments may also be synthesized chemically; thus,the smallest toxic fragment obtainable from transcription andtranslation of synthetic DNA is included in the definition of smallesttoxic fragment.

In one embodiment of the invention, the smallest toxic fragment of anISP3 protein has a molecular weight of about 65 kDa as determined bySDS-PAGE (Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis)analysis. In another embodiment, the smallest toxic fragment of an ISP3protein has a molecular weight of about 23 kDa. In another embodiment,the smallest toxic fragment of an ISP3 protein has a molecular weight ofabout 33 kDa. In a further embodiment, the smallest toxic fragmentcomprises the central amino acids of the ISP3 protein, in particularfrom amino acid 200 to amino acid 455 of SEQ ID NO: 2, SEQ ID NO: 4 orSEQ ID NO: 6.

Enzymatic digestions of ISP3 proteins can be performed by using purifiedenzymes, or by using gut juice fluids from insect larvae and incubatinggut juice extracts with solutions comprising one of the ISP3 proteins,as described in Yu et al. (1997). Proteolytic products can be separatedand visualized on SDS-PAGE. Bioassays can be carried out with processed,chromatographically fractioned protein fragments in order to determinethe relationship between each proteolytic fragment and its insecticidalactivity. Gut juice used to determine the smallest toxic fragment ofISP3 proteins may be gut juice from Lepidopteran insects. SuitableLepidopteran insects include, but are not limited to, Corn Earworm(Helicoverpa zea), Cotton Bollworm (Helicoverpa armigera), NativeBudworm (Helicoverpa punctigera), Tobacco Budworm (Heliothis virescens),European Corn Borer (Ostrinia nubilalis), Fall Armyworm (Spodopterafrugiperda), Black Cutworm (Agrotis ipsilon), Pink Bollworm(Pectinophora gossypiella), Yellow Stem Borer (Scirphophaga incertulas),Leaffolder (Cnaphalocrocis medinalis), Pink Stem Borer (Sesamiainferens), Corn Spotted Stem Borer (Chilo partellus), Velvet Caterpillar(Anticarsia gemmatalis), Soybean Looper (Pseudoplusia includens), PodBorer (Epinotia aporema), and Rachiplusia nu.

The N- and C-terminal amino acid sequences of the smallest toxicfragment may be conveniently determined by amino acid sequencedetermination of the above fragments using routine techniques availablein the art.

As used herein, the terms “isp3-1099E”, “isp3-327D” and “isp3-2245J”refer to any DNA sequence encoding the “ISP3-1099E protein” or“ISP3-327D protein” or “ISP3-2245J protein”, respectively, as definedabove. This includes naturally-occurring, artificial, or synthetic DNAsequences encoding the proteins of SEQ ID NO: 2 or SEQ ID NO: 4 or SEQID NO: 6, or their insecticidal fragments or variants as defined above.Also included herein are DNA sequences encoding insecticidal proteins,which are similar enough to the DNA sequences provided in the sequencelisting that they can (i.e., have the ability to) hybridize to these DNAsequences under stringent hybridization conditions.

“Stringent hybridization conditions”, as used herein, refersparticularly to the following conditions: immobilizing the relevant DNAon a filter, and prehybridizing the filters for either 1 to 2 hours in50% formamide, 5×SSPE, 2×Denhardt's reagent and 0.1% SDS at 42° C., or 1to 2 hours in 6×SSC, 2×Denhardt's reagent and 0.1% SDS at 68° C. Thedenatured (Digoxigenin- or radio-) labeled probe is then added directlyto the prehybridization fluid and incubation is carried out for 16 to 24hours at the appropriate temperature mentioned above. After incubation,the filters are then washed for 30 minutes at room temperature in 2×SSC,0.1% SDS, followed by 2 washes of 30 minutes each at 68° C. in 0.5×SSCand 0.1% SDS. An autoradiograph is established by exposing the filtersfor 24 to 48 hours to X-ray film (Kodak XAR-2 or equivalent) at −70° C.with an intensifying screen (20×SSC=3M NaCl and 0.3M sodium citrate;100×Denhardt's reagent=2% (w/v) bovine serum albumin, 2% (w/v) FicoII™and 2% (w/v) polyvinylpyrrolidone; SDS=sodium dodecyl sulfate;20×SSPE=3.6M NaCl, 02M Sodium phosphate and 0.02M EDTA pH7.7). One ofordinary skill in the art will readily be able to modify the particularconditions and parameters specified above while retaining the desiredstringent hybridization conditions.

There are many approaches known in the art for the isolation of variantsof the DNA sequences of the invention. For example, variants can beisolated from Bt strains by hybridization as described supra, and/or byPCR technology, as known in the art. Specific or degenerate primers canbe made to regions of the isp3 DNA sequences, and used to amplifyvariants from known or novel Bt strains.

Variants of the isp3-1099E DNA of the invention include DNA sequencesencoding the insecticidal ISP3-1099E protein variants described above,or a DNA sequence, encoding an insecticidal protein, with at least 93%,at least 94%, at least 95%, 96% or 97%, at least 98% or at least 99%sequence identity to SEQ ID NO: 1. Variants of the isp3-327D DNA of theinvention are DNA sequences encoding the ISP3-327D protein variantsdescribed above, or a DNA sequence, encoding an insecticidal protein,with at least 94%, at least 95%, at least 96%, 97%, 98% or at least 99%sequence identity to SEQ ID NO: 3. Variants of the isp3-2245J DNA ofthis invention are DNA sequences encoding the ISP3-2245J proteinvariants described above, or a DNA sequence, encoding an insecticidalprotein, with at least 97%, at least 98%, or at least 99% sequenceidentity to SEQ ID NO: 5. The sequence identities referred to above arecalculated using the GAP program of the Wisconsin package of GCG(Madison, Wis., USA) Version 10.2. The GAP program is used with thefollowing parameters for nucleic acids: the “nwsgapdna” scoring matrix,a “gap creation penalty” (or “gap weight”) of 50 and a “gap extensionpenaltc(or “length weight”) of 3. Stringent hybridization conditions areas defined above.

“Insecticidal activity” of a protein, as used herein, means the capacityof a protein to kill insects when such protein is fed to insects,preferably by expression in a recombinant host such as a plant. It isunderstood that a protein has insecticidal activity if it has thecapacity to kill the insect during at least one of its developmentalstages, preferably the larval stage.

“Insect-controlling amounts” of a protein, as used herein, refers to anamount of protein which is sufficient to limit damage on a plant, causedby insects at any stage of development (e.g. insect larvae) feeding onsuch plant, to commercially acceptable levels. Limiting insect damage toa plant may be the result of, for example, killing the insects orinhibiting insect development, fertility or growth in such a manner thatthe insect inflicts less damage to a plant and plant yield is notsignificantly adversely affected.

In accordance with this invention, insects susceptible to the new ISP3proteins of the invention are contacted with this protein ininsect-controlling amounts, preferably insecticidal amounts. Preferredtarget insects for the proteins of this invention are economicallydamaging insect pests of corn, cotton, rice or soybean plants,particularly in Northern and Southern American countries, Asia andAustralia. The term plant, as used herein, encompasses whole plants aswell as parts of plants, such as leaves, stems, seeds, flowers or roots.Target insects for the ISP3 proteins of this invention include, but arenot limited to, lepidopteran insect pests, such as Heliothis spp.,Helicoverpa spp., Spodoptera spp., Ostrinia spp., Pectinophora spp,Agrotis spp., Scirphophaga spp., Cnaphalocrocis spp., Sesamia spp, Chilospp., Anticarsia spp., Pseudoplusia spp., Epinotia spp., and Rachiplusiaspp., preferably Heliothis virescens, Helicoverpa zea, Helicoverpaarmigera, Helicoverpa punctera, Ostrinia nubilalis, Spodopterafrugiperda, Agrotis ipsilon, Pectinophora gossypiella, Scirphophagaincertulas, Cnaphalocrocis medinalis, Sesamia inferens, Chilo partellus,Anticarsia gemmatalis, Pseudoplusia includens, Epinotia aporema andRachiplusia nu. The ISP3 proteins of the invention may have insecticidalactivity against at least one lepidopteran insect species, and may haveactivity against several Lepidopteran insect species.

The terms “ISP3 protein”, “ISP3 protein of this invention”, “ISPprotein”, or “ISP protein of this invention”, as used herein, refers toany one of the new proteins isolated in accordance with this invention,and identified and defined herein as ISP3-1099E, ISP3-327D, orISP3-2245J protein.

An ISP3 protein, as used herein, can be a protein in the full-lengthsize or can be in a truncated form as long as the insecticidal activityis retained, or can be a combination of different proteins or proteindomains in a hybrid or fusion protein. An “ISP3 toxin” refers to aninsecticidal fragment or portion of an ISP3 protein, particularly thesmallest toxic fragment thereof. An “isp gene,” “isp3 gene,” “isp DNA,”or “isp3 DNA,” as used herein, is a DNA sequence encoding an ISP3protein in accordance with this invention, referring particularly to anyof the above-defined isp3-1099E, isp3-327D or isp3-2245J DNA sequences.

The nucleic acid sequence, particularly DNA sequence, encoding the ISP3proteins of this invention can be made synthetically and can be insertedin expression vectors to produce high amounts of ISP3 proteins. The ISP3proteins can be used to prepare specific monoclonal or polyclonalantibodies in a conventional manner (Höfte et al., 1988; Harlow andLane, 1988).

In one embodiment of the invention, antibodies that specifically bind tothe ISP3 protein are provided. In particular, monoclonal or polyclonalantibodies that bind to ISP3-1099E, ISP3-327D or ISP3-2245J or tofragments or variants thereof are provided. Also included are fragmentsof monoclonal or polyclonal antibodies, which retain the ability to bindto the ISP3 protein or fragment against which they were raised. Anantibody to an ISP3 protein can be prepared by using the ISP3 protein asan antigen in an animal (such as rabbit or mouse), using methods knownin the art. Suitable methods for preparing antibodies include thosedescribed in Harlow and Lane “Using Antibodies: A Laboratory Manual”(New York: Cold Spring Harbor Laboratory Press, 1998); and in Liddelland Cryer “A Practical Guide to Monoclonal Antibodies” (Wiley and Sons,1991). The antibodies can be used to isolate, identify, characterize orpurify the ISP3 protein to which it binds. For example, the antibody canbe used to detect the ISP3 protein in a sample, by allowing antibody andprotein to form an immunocomplex, and detecting the presence of theimmunocomplex, for example through ELISA or immunoblots.

In addition, immunological kits, useful for the detection of ISP3proteins, protein fragments or epitopes in a sample are provided.Samples may be cells, cell supernatants, cell suspensions, and the like.Such kits comprise an antibody that binds to the ISP3 protein (orfragment thereof) and one or more immunodetection reagents.

The antibodies can also be used to isolate insecticidal proteins withsimilar activity by for example ELISA (Enzyme Linked Immuno-SorbentAssay) or Western blotting. Monoclonal antibody lines with desiredbinding specificity can also be used to clone the DNA for the particularmonoclonal antibody.

In a further embodiment of the invention PCR primers and/or probes andkits for detecting the isp3-1099E, isp3-237D or isp3-2245J DNA sequencesare provided. PCR primer pairs to amplify isp3 DNA from samples can besynthesized based on SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, asknown in the art (see, e.g., Dieffenbach and Dveksler (1995) PCR Primer:A Laboratory Manual, Cold Spring Harbor Laboratory Press; and McPhersonat al. (2000) PCR—Basics: From Background to Bench, First Edition,Springer Verlag, Germany). Likewise, DNA fragments of SEQ ID NO: 1, SEQID NO: 3 or SEQ ID NO: 5 can be used as hybridization probes. An isp3detection kit may comprise either isp3 specific primers or isp3 specificprobes, and an associated protocol to use the primers or probe to detectisp3 DNA in a sample. For example, such a detection kit may be used todetermine, whether a plant has been transformed with an isp3 gene (orpart thereof) of the invention.

Because of the degeneracy of the genetic code, some amino acid codonscan be replaced by others without changing the amino acid sequence ofthe protein. Furthermore, some amino acids can be substituted by otherequivalent amino acids without changing, or without significantlychanging the insecticidal activity of the protein, or at least withoutchanging the insecticidal activity of the protein in a negative way. Forexample, conservative amino acid substitutions include interchangingamino acids within categories: basic (e.g. Arg, His, Lys), acidic (e.g.Asp, Glu), nonpolar (e.g. Ala, Val, Trp, Leu, Ile, Pro, Met, Phe, Trp),and polar (e.g. Gly, Ser, Thr, Tyr, Cys, Asn, Gln). Such substitutionswithin categories fall within the scope of the invention as long as theinsecticidal activity of the ISP3 protein is not changed, or notsignificantly changed, or at least not changed in a negative way. Inaddition, non-conservative amino acid substitutions fall within thescope of the invention as long as the insecticidal activity of the ISP3protein is not changed, or not significantly changed, or at least is notchanged in a negative way. Variants or equivalents of the DNA sequencesof the invention include DNA sequences hybridizing to the isp3 DNAsequence of SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 5 under stringenthybridization conditions. Such variants or equivalents will encode aprotein with the same insecticidal characteristics as the protein ofthis invention. Variants or equivalents also include DNA sequenceshaving a different codon usage compared to the native isp3 genes of thisinvention but which encode a protein with the same insecticidal activityand with the same or substantially the same amino acid sequence. Theisp3 DNA sequences can be codon-optimized by adapting the codon usage tothat most preferred in plant genes, particularly to genes native to theplant genus or species of interest (Bennetzen & Hall, 1982; Itakura etal., 1977) using available codon usage tables (e.g. more adapted towardsexpression in cotton, soybean corn or rice). Codon usage tables forvarious plant species have been published by, for example, Ikemura(1993) and Nakamura et al. (2000).

Long stretches of AT or GC nucleotides may be removed and suitablerestriction sites may be introduced. In addition, the N-terminus of anISP3 protein can be modified to have an optimum translation initiationcontext, thereby adding, replacing or deleting one or more amino acidsat the N-terminal of the protein. In most cases, it is preferred thatthe proteins of the invention to be expressed in plants cells start witha Met-Asp or Met-Ala dipeptide for optimal translation initiation,requiring the insertion in the isp3 DNA of a codon encoding an Asp orAla amino acid downstream of the start codon as a new second codon.Alternatively, the fourth nucleotide of SEQ ID NO: 1, SEQ ID NO: 3 orSEQ ID NO: 5 may be replaced by a “G,” so that the second amino acid(following Met) is Asp. Likewise, the second codon (AAC or AAT, codingfor Asn) may be replaced by a codon for Asp (GAT or GAC), or Ala (GCT,GCC, GCA or GCG), or by any other codon starting with a “G.”

The DNA sequences may also be modified to remove illegitimate splicesites. As bacterial genes may contain motifs that are recognized inother hosts, especially in eukaryotic host such as plants, as 5′ or 3′splice sites, transcription in those other hosts may be terminatedprematurely, resulting in truncated mRNA. Illegitimate splice sites canbe identified by computer-based analysis of the DNA sequences and/or byPCR analysis as known in the art.

Any DNA sequence differing in its codon usage but encoding the sameprotein or a similar protein with substantially the same insecticidalactivity can be constructed, depending on the particular purpose. It hasbeen described in prokaryotic and eukaryotic expression systems thatchanging the codon usage to that of the host cell has benefits for geneexpression in foreign hosts (Bennetzen & Hall, 1982; Itakura et al.,1977). Codon usage tables are available in the literature (Wada et al.,1990; Murray et al., 1989) and in the major DNA sequence databases (e.g.EMBL at Heidelberg, Germany) and as described by Nakamura et al (2000).Accordingly, one of ordinary skill in the art can readily constructsynthetic DNA sequences so that the same or substantially the sameproteins are produced. It is evident that alternate DNA sequences can bemade once the amino acid sequence of the ISP3 proteins of this inventionis known. Such alternate DNA sequences include synthetic orsemi-synthetic DNA sequences that have been changed in order toinactivate certain sites in the gene. This inactivation can beaccomplished by, for example, selectively inactivating certain crypticregulatory or processing elements present in the native sequence asdescribed in PCT publications WO 91/16432 and WO 93/09218, or adaptingthe overall codon usage to that of a more related host organism, such asthat of the host organism in which expression is desired. Severaltechniques for modifying the codon usage to that preferred by the hostcells can be found in the patent and scientific literature. The exactmethod of codon usage modification is not critical for this invention aslong as most or all of the cryptic regulatory sequences or processingelements have been replaced by other sequences.

Small modifications to a DNA sequence such as described above can beroutinely made, e.g., by PCR-mediated mutagenesis (Ho et al., 1989,White et al., 1989). Greater modifications to a DNA sequence canroutinely be made by de novo DNA synthesis of a desired coding regionusing available techniques.

The phrase “substantially the same,” when used herein in reference tothe amino acid sequence of an ISP3 protein, refers to an amino acidsequence that differs no more than 5%, or no more than 2%, from theamino acid sequence of the protein compared to. When referring totoxicity of an ISP3 protein, the phrase “substantially the same” refersto a protein whose mean LC₅₀ value differs by no more than a factor of 2from the mean LC₅₀ value obtained for the protein compared to. In thiscontext, “mean LC₅₀” is the concentration of protein causing 50%mortality of the test population, calculated from three independentbioassays carried out using the same bioassay conditions. LC₅₀ valuesare calculated with Probit analysis, using the program POLO PC (fromLeOra Software, 1987, Berkely, Calif.). It is understood, that 95% (or90%) confidence limits (an associated parameter calculated with Probitanalysis) are calculated for the LC₅₀ values of each of the two proteinsto be compared in order to determine whether a statistically significantdifference in LC₅₀ values exists. In general, the toxicity of the twoproteins is seen to be substantially the same, if the confidence limitsoverlap and substantially different if the confidence limits do notoverlap.

The term “domain” of a ISP3 toxin (or ISP3 protein) as used herein meansany part(s) of the toxin (or ISP3 protein) with a specific structure orfunction that can be transferred to another protein for providing a newhybrid protein with at least one functional characteristic (e.g., thebinding and/or toxicity characteristics) of the ISP3 toxin (or ISP3protein) of the invention (Ge et al., 1991). Such parts can form anessential feature of the hybrid protein with the binding and/or toxicitycharacteristics of the ISP3 proteins of this invention. Such a hybridprotein can have an enlarged host range, an improved toxicity and/or canbe used in a strategy to prevent insect resistance development (EP 408403; Visser et al., 1993). DNA sequences encoding the domains areencompassed by this definition. A hybrid protein or fusion protein isused herein to mean a protein comprised of different protein domains,forming a functional, chimeric protein with the characteristics of theindividual domains. Another domain which a hybrid or chimeric proteinmay, for example, comprise is a stabilizing domain. Stabilizing domainshave for example been described to be present at the C-terminus ofVIP3(a) proteins, and are thought to provide stability to the toxicprotein in the gut-environment of susceptible insects.

In addition to creating hybrid proteins, the function of specificdomains can also be analyzed by the introduction of deletions of all orpart of the domain(s) or the introduction of mutations into the domain,and analysis of the resulting effect on toxicity towards insects,protein stability, sensitivity to enzyme proteolysis, temperaturechanges, binding to DNA/proteins/specific cells, etc.

The present invention also provides a method of “evolving” a nucleicacid sequence encoding an ISP3 protein, particularly ISP3-1099E orISP3-327D or ISP32245J, into a new nucleic acid sequence that encodes aprotein having insecticidal activity. The evolved nucleic acid sequencecan have improved insecticidal activity compared to the non-evolvedsequence. The term “evolving” as used herein refers to a method ofenhanced sequence evolution by recombination of sequences, as describedin U.S. Pat. No. 5,811,238, WO97/20078 and U.S. Pat. No. 6,180,406,incorporated herein by reference. Nucleic acid “shuffling” is usedherein to indicate in vitro or in vivo recombination between nucleicacid sequences of a nucleic acid population or pool and can be carriedout as known in the art and as described in U.S. Pat. No. 5,811,238,WO97/20078, U.S. Pat. No. 6,180,406, U.S. Pat. No. 6,117,679, allincorporated herein by reference.

The method of evolving a nucleic acid sequence encoding an ISP3 proteincomprises the following steps:

-   a) providing a population of nucleic acid sequences encoding the    amino acid sequences of SEQ ID NO: 2 and/or SEQ ID NO: 4 and/or SEQ    ID NO: 6, or variants or fragments of the amino acid sequences of    SEQ ID NO: 2 and/or SEQ ID NO: 4 and/or SEQ ID NO: 6, wherein said    variants or fragments have a sequence identity of at least 91% to    SEQ ID NO: 2 or SEQ ID NO: 4 and at least 88% to SEQ ID NO: 6;-   b) shuffling said population of variants or fragments to form    recombinant nucleic acid molecules;-   c) selecting or screening for recombinant nucleic acid molecules,    which encode proteins that have insecticidal activity; and-   d) repeating steps (a) to (c) with the recombinant nucleic acid    molecules selected in step (c) until a recombinant nucleic acid    molecule has been found in step (c), wherein the protein encoded by    said nucleic acid molecule has the desired insecticidal property.

A non-evolved nucleic acid is a nucleic acid provided as startingmaterial in step (a), while a corresponding evolved nucleic acid as usedherein refers to a recombinant nucleic acid obtained in step (d) whencarrying out the method using the non-evolved nucleic acid in step (a).Preferred nucleic acids used in step (a) are nucleic acid sequencesencoding amino acid sequences ISP3-1099E (SEQ ID NO: 2) and/or ISP3-327D(SEQ ID NO: 4) and/or ISP3-2245J (SEQ ID NO: 6) or variants or fragmentsthereof. The population of nucleic acid molecules and/or variants and/orfragments of nucleic acid molecules in step (a) may comprise the DNAencoding a single ISP3 protein and/or variants and/or fragments of thenucleic acid encoding a single ISP3 protein of the invention, or amixture of nucleic acids encoding different ISP3 proteins of theinvention, and/or fragments and/or variants thereof. Nucleic acidsequences encoding variants of amino acid sequence SEQ ID NO: 2 arenucleic acid sequences encoding amino acid sequences which have at least91%, at least 92%, at least 93%, 94%, 95%, 98%, 99% or 100% sequenceidentity at the amino acid level to SEQ ID NO: 2. Nucleic acid sequencesencoding variants of amino acid sequence SEQ ID NO: 4 are nucleic acidsequences encoding amino acid sequences which have at least 91%, atleast 92 or 93%, at least 94%, 95%, 98%, 99% or 100% sequence identityat the amino acid level to SEQ ID NO: 4. Nucleic acid sequences encodingvariants of amino acid sequence SEQ ID NO: 6 are nucleic acid sequencesencoding amino acid sequences which have at least 88%, at least 89 or90%, at least 91%, 92%, 93%, 95%, 98%, 99% or 100% sequence identity atthe amino acid level to SEQ ID NO: 6.

An evolved nucleic acid sequence obtained in step (d) may encode aprotein with improved insecticidal activity. Such a protein has, forexample, either higher toxicity than the protein encoded by thenon-evolved sequences used in step (a) as starting material, or hasactivity against a different spectrum of target insects than thenon-evolved sequences, or it binds to a different target binding site ina target insect. Selection or screening for the desired higher toxicityand/or different toxicity spectrum (step (c)) can be carried out byperforming insect bioassays, comparing insecticidal activity of theproteins encoded by the evolved and non-evolved nucleic acid sequences.Alternative or additional other functional assays may be carried out,depending on the desired insecticidal property. For example, if enhancedbinding is a desired property, a binding assay may be carried out priorto carrying out an insect bioassay.

The isp3 DNA sequences of the invention, prepared from total DNA, can beligated in suitable expression vectors and transformed in a bacterialstrain, such as E. coli or a Bt strain. The clones can then be screenedby conventional colony immunoprobing methods (French et al., 1986) forexpression of the toxin with monoclonal or polyclonal antibodies raisedagainst the ISP3 proteins. The Bt or E. coli clones can be screened forproduction of ISP3 proteins (cell-free culture supernatant or celllysate can be run on SDS-PAGE gels using standard methods and standardwestern-blotting procedures can be carried out), or the bacteria can betested for their insecticidal activity compared to the control bacteria.The clones can also be analysed for the presence of mRNA encoding ISP3protein using standard PCR procedures, such as RT-PCR.

The genes encoding the ISP3 proteins of this invention can be sequencedin a conventional manner (Maxam and Gilbert, 1980; Sanger, 1977) toobtain the DNA sequence. Sequence comparisons indicated that the genesare different from previously described genes encoding toxins with,activity against Lepidoptera.

An insecticidally-effective part of the DNA sequences, encoding aninsecticidally-effective portion of the newly identified ISP3 proteins,can be made in a conventional manner after sequence analysis of thegene. The amino acid sequence of the ISP3 proteins can be determinedfrom the DNA sequence of the isolated DNA sequences. The phrase “aninsecticidally effective part (or portion or fragment)” of DNA sequencesencoding the ISP3 protein, also referred to herein as a “truncated gene”or “truncated DNA,” as used herein refers to a DNA sequence encoding apolypeptide that is insecticidal, but has fewer amino acids than theISP3 full length protein form.

In order to express all or an insecticidally-effective part of the DNAsequence encoding an ISP3 protein of this invention in E. coli, in otherBt strains, or in plants, suitable restriction sites can be introduced,flanking the DNA sequence. This can be done by site-directedmutagenesis, using well-known procedures (see, e.g., Stanssens et al.,1989; White et al., 1989). In order to obtain improved expression inplants, the codon usage of the isp3 gene or insecticidally effectiveisp3 gene part of this invention can be modified to form an equivalent,modified or artificial gene or gene part in accordance with PCTpublications WO 91/16432 and WO 93/09218 and publications EP 0 385 962,EP 0 359 472 and U.S. Pat. No. 5,689,052. The isp3 genes or gene partsmay also be inserted in the plastid, mitochondrial or chloroplast genomeand expressed there using a suitable promoter (see, e.g., Mc Bride etal., 1995; U.S. Pat. No. 5,693,507).

For obtaining enhanced expression in monocot plants such as corn orrice, an intron (e.g., a monocot intron) can also be added to thechimeric gene. For example, the insertion of the intron of the maizeAdhl gene into the 5′ regulatory region has been shown to enhanceexpression in maize (Callis et. al., 1987). Likewise, the HSP70 intron,as described in US 5,859,347, may be used to enhance expression. The DNAsequence of the isp3 gene or its insecticidal part can be furtherchanged in a translationally neutral manner. Such changes may modifypossibly inhibiting DNA sequences present in the gene part by means ofsite-directed intron insertion and/or by introducing changes to thecodon usage. Changes in codon usage may, e.g., adapt the codon usage tothat most preferred by plants, such as the specific relevant plant genus(Murray et al., 1989), without changing, or without significantlychanging, the encoded amino acid sequence.

In accordance with one embodiment of this invention, the proteins may betargeted to intracellular organelles such as plastids, chloroplasts,mitochondria, or are secreted from the cell, potentially optimizingprotein stability and/or expression. For this purpose, in one embodimentof this invention, the chimeric genes of the invention comprise a codingregion encoding a signal or target peptide, linked to the ISP3protein-coding region of the invention. Peptides that may be included inthe proteins of this invention are the transit peptides for chloroplastor other plastid targeting, such as (duplicated) transit peptide regionsfrom plant genes whose gene product is targeted to the plastids, theoptimized transit peptide of Capellades et al. (U.S. Pat. No.5,635,618), the transit peptide of ferredoxin-NADP⁺ oxidoreductase fromspinach (Oelmuller et al., 1993), the transit peptide described in Wonget al. (1992) and the targeting peptides in published PCT patentapplication WO 00/26371. Alternative peptides include those signallingsecretion of a protein linked to such peptide outside the cell, such asthe secretion signal of the potato proteinase inhibitor II (Keil et al.,1986), the secretion signal of the alpha-amylase 3 gene of rice (Sutliffet al., 1991) and the secretion signal of tobacco PR1 protein(Cornelissen et al., 1986).

Useful signal peptides in accordance with the invention include thechloroplast transit peptide (e.g., Van Den Broeck et al., 1985), or theoptimized chloroplast transit peptide of U.S. Pat. No. 5,510,471 andU.S. Pat. No. 5,635,618 causing transport of the protein to thechloroplasts, a secretory signal peptide or a peptide targeting theprotein to other plastids, mitochondria, the ER, or another organelle.Signal sequences for targeting to intracellular organelles or forsecretion outside the plant cell or to the cell wall are found innaturally targeted or secreted proteins, such as those described byKlösgen et al. (1989), Klösgen and Weil (1991), Neuhaus & Rogers (1998),Bih et al. (1999), Morris et al. (1999), Hesse et al. (1989),Tavladoraki et al. (1998), Terashima et al. (1999), Park et al. (1997),Shcherban et al. (1995), all of which are incorporated herein byreference. Alternative signal sequences include the signal peptidesequences from targeted or secreted proteins of corn, cotton, soybean orrice.

To allow secretion of the ISP3 proteins to the outside of thetransformed host cell, an appropriate secretion signal peptide may befused to the amino terminal end (N-terminal end) of the ISP3 protein.Also, any putative native Bacillus secretion signal peptide can bedeleted or can be replaced by an appropriate signal peptide, such as aeukaryotic secretion signal peptide as described above. Particularly,amino acids 1 to 54 of the ISP3 proteins of the invention comprise aputative Bacillus signal peptide. Amino acids 1 to 10, 1 to 50, or 1 to54 may be removed from the ISP3 protein or may be replaced by anappropriate signal peptide, such as a eukaryotic signal peptide asdescribed above. Putative signal peptides can be detected using computerbased analysis, using programs such as the program Signal Peptide search(SignalP V1.1 or 2.0), using a matrix for prokaryotic gram-positivebacteria and a threshold score of less than 0.5, a threshold score of0.25, or less (see, e.g., Von Heijne, Gunnar, 1986 and Nielsen et al.,1996).

Furthermore, the binding properties of the ISP3 proteins of theinvention can be evaluated, using methods known in the art (see, e.g.,Van Rie et al., 1990), to determine if the ISP3 proteins of theinvention bind to sites in the insect gut, such as the midgut, that arenot recognized (or competed for) by other Bt proteins. Insecticidal Btproteins with different binding sites (for which there is no competitionfor binding) in relevant susceptible insects are very valuable. Suchproteins can be used to replace known Bt proteins to which insects mayhave developed resistance, or to use in combination with insecticidal Btproteins having a different mode of action to prevent or delay thedevelopment of insect resistance against Bt proteins, particularly whenexpressed in a plant. Because of the characteristics of the Bt toxins ofthe present invention, they are extremely useful for transformingplants, e.g. monocots such as corn and rice and dicots such as cottonand soybean, to protect these plants from insect damage. It is expectedthat the binding properties of the ISP3 proteins of the currentinvention will be different compared to those of Cry toxins. Suchdifferent binding properties can be measured by routine binding assaysas described above or in U.S. Pat. No. 6,291,156 and U.S. Pat. No.6,137,033.

Especially for insect resistance management purposes for a specificinsect pest, it is preferred to combine an ISP3 protein of thisinvention with another insect control protein, particularly a Bt Cryprotein or a VIP or VIP-like protein, preferably a protein which doesnot recognise at least one binding site recognised by such ISP3 protein.Suitable insect control proteins to combine with the ISP3 proteins ofthis invention, particularly for simultaneous expression in plants (suchas maize, cotton, rice or soybean plants), include, but are not limitedto, the Cry proteins, such as the Cry1F protein or hybrids derived froma Cry1F protein (e.g., the hybrid Cry1A-Cry1F proteins described in U.S.Pat. No. 6,326,169; U.S. Pat. No. 6,281,016; U.S. Pat. No. 6,218,188, ortoxic fragments thereof), the Cry1A-type proteins or toxic fragmentsthereof, preferably the Cry1Ac protein or hybrids derived from theCry1Ac protein (e.g., the hybrid Cry1Ab-Cry1Ac protein described in U.S.Pat. No. 5,880,275) or the Cry1Ab or Bt2 protein or insecticidalfragments thereof as described in EP451878, the Cry2Ae, Cry2Af or Cry2Agproteins as described in WO02/057664, the Cry proteins as described inWO01/47952, the VIP3Aa protein or a toxic fragment thereof as describedin Estruch et al. (1996) and U.S. Pat. No. 6,291,156, insecticidalproteins from Xenorhabdus (as described in WO98/50427), Serratia(particularly from S. entomophila) or Photorhabdus species strains, suchas Tc-proteins from Photorhabdus as described in WO98/08932 (e.g.,Waterfield et al., 2001; Ffrench-Constant and Bowen, 2000). In oneembodiment, such co-expression is easily obtained by transforming aplant already expressing an insect control protein with a ISP3 of thisinvention, or by crossing plants transformed with the insect controlprotein and plants transformed with one or more ISP proteins of thisinvention. For maize, rice, cotton or soybean plants, the ISP3-327Dprotein or the ISP3-1099E protein or the ISP3-2245J protein may be usedas first insect control protein and as second insect control protein theCry1Ab, Cry1Ac, Cry2Ae or VIP3Aa proteins or derivatives thereof can beused. Methods for obtaining expression of different Bt (or similarly,for other insect control proteins) insecticidal proteins in the sameplant in an effort to minimize or prevent resistance development totransgenic insect-resistant plants are described in EP 0 408 403. Thedifferent proteins can be expressed in the same plant, or each can beexpressed in a single plant and then combined in the same plant bycrossing the single plants with one another. For example, in hybrid seedproduction, each parent plant can express a single protein. Uponcrossing the parent plants to produce hybrids, both proteins arecombined in the hybrid plant.

It is well known that Bt Cry proteins are expressed as protoxins, whichare converted into the toxic core by proteolysis in the insect gut. Whencombining the ISP3 proteins of the invention with Bt Cry proteins, it isunderstood that Bt Cry genes encoding either the full protoxin or thetoxic core or any intermediate form may be used.

For selection purposes, and for increasing the weed control options, thetransgenic plants of the invention may also be transformed with a DNAencoding a protein conferring resistance to a broad-spectrum herbicide,e.g., herbicides based on glufosinate or glyphosate.

The insecticidally effective isp3 gene part or its equivalent,preferably the isp3 chimeric gene, encoding an insecticidally effectiveportion of the ISP3 protein, can be stably inserted in a conventionalmanner into the nuclear genome of a single plant cell, and theso-transformed plant cell can be used in a conventional manner toproduce a transformed plant that is insect-resistant. In this regard, aT-DNA vector, containing the insecticidally effective isp3 gene part, inAgrobacterium tumefaciens can be used to transform the plant cell.Thereafter, a transformed plant can be regenerated from the transformedplant cell using the procedures described, for example, in EP 0 116 718,EP 0 270 822, PCT publication WO 84/02913 and published European Patentapplication EPO 242 246 and in Gould et al. (1991). The construction ofa T-DNA vector for Agrobacterium-mediated plant transformation is wellknown in the art. The T-DNA vector may be either a binary vector asdescribed in EP 0 120 561and EP 0 120 515 or a co-integrate vector whichcan integrate into the Agrobacterium Ti-plasmid by homologousrecombination, as described in EP 0 116 718. Preferred T-DNA vectorseach contain a promoter operably linked to the insecticidally effectiveisp3 gene part between T-DNA border sequences, or at least located tothe left of the right border sequence. Border sequences are described inGielen et al. (1984). Other types of vectors can be used to transformthe plant cell, using procedures such as direct gene transfer (asdescribed, for example in EP 0 223 247), pollen mediated transformation(as described, for example in EP 0 270 356 and WO 85/01856), protoplasttransformation as, for example, described in U.S. Pat. No. 4,684,611,plant RNA virus-mediated transformation (as described, for example in EP0 067 553 and U.S. Pat. No. 4,407,956), liposome-mediated transformation(as described, for example in U.S. Pat. No. 4,536,475), and othermethods, such as the recently described methods for transforming certainlines of corn (e.g., U.S. Pat. No. 6,140,553; Fromm et al., 1990;Gordon-Kamm et al., 1990) and rice (Shimamoto et al., 1989; Datta et al.1990) and the method for transforming monocots generally (PCTpublication WO 92/09696). A suitable method for cotton transformation isdescribed in PCT patent publication WO 00/71733. For ricetransformation, reference is made to the methods described inWO92/09696, WO94/00977 and WO95/06722.

The terms “maize” and “corn” are used herein synonymously, referring toZea mays. Cotton as used herein refers to Gossypium spp., particularlyG. hirsutum and G. barbadense. The term “rice” refers to Oryza spp.,particularly O. sativa. “Soybean” refers to Glycine spp, particularly G.max.

Besides transformation of the nuclear genome, also transformation of theplastid genome (e.g., the chloroplast genome) is included in theinvention. Kota et al. (1999) have described a method to over-express aCry2Aa protein in tobacco chloroplasts.

The resulting transformed plant can be used in a conventional plantbreeding scheme to produce more transformed plants with the samecharacteristics or to introduce the insecticidally effective isp3 genepart into other varieties of the same or related plant species. Seeds,which are obtained from the transformed plants, contain theinsecticidally effective isp3 gene part as a stable genomic insert.Cells of the transformed plant can be cultured in a conventional mannerto produce the insecticidally effective portion of the ISP3 toxin orprotein, which can be recovered for use in conventional insecticidecompositions against Lepidoptera (see, e.g., U.S. Pat. No. 5,254,799).

The insecticidally effective isp3 gene part is inserted in a plant cellgenome so that the inserted gene is downstream (i.e., 3′) of, and underthe control of, a promoter which can direct the expression of the genepart in the plant cell. This may be accomplished by inserting the isp3chimeric gene in the plant cell genome, for example in the nuclear orplastid (e.g., chloroplast) genome.

Suitable promoters include, but are not limited to: the strongconstitutive 35S promoters (the “35S promoters”) of the cauliflowermosaic virus (CaMV) of isolates CM 1841 (Gardner et al., 1981), CabbB-S(Franck et al., 1980) and CabbB-JI (Hull and Howell, 1987); the 35Spromoter described by Odell et al. (1985), promoters from the ubiquitinfamily (e.g., the maize ubiquitin promoter of Christensen et al., 1992,EP 0 342 926, see also Cornejo et al., 1993), the gos2 promoter (dePater et al., 1992), the emu promoter (Last et al., 1990), Arabidopsisactin promoters such as the promoter described by An et al. (1996), riceactin promoters such as the promoter described by Zhang et al. (1991)and the promoter described in U.S. Pat. No. 5,641,876; promoters of theCassava vein mosaic virus (WO 97/48819, Verdaguer et al. (1998)), thepPLEX series of promoters from Subterranean Clover Stunt Virus (WO96/06932, particularly the S7 promoter), a alcohol dehydrogenasepromoter, e.g., pAdh1S (GenBank accession numbers X04049, X00581), andthe TR1′ promoter and the TR2′ promoter (the “TR1′ promoter” and “TR2′promoter”, respectively) which drive the expression of the 1′ and 2′genes, respectively, of the T-DNA (Velten et al., 1984). Alternatively,a promoter can be utilized which is not constitutive but rather isspecific for one or more tissues or organs of the plant (e.g., leavesand/or roots) whereby the inserted isp3 gene part is expressed only incells of the specific tissue(s) or organ(s). For example, theinsecticidally effective isp3 gene part could be selectively expressedin the leaves of a plant (e.g., corn, cotton, rice, soybean) by placingthe insecticidally effective gene part under the control of alight-inducible promoter such as the promoter of theribulose-1,5-bisphosphate carboxylase small subunit gene of the plantitself or of another plant, such as pea, as disclosed in U.S. Pat. No.5,254,799. The promoter can, for example, be chosen so that the isp3gene of the invention is only expressed in those tissues or cells onwhich the target insect pest feeds so that feeding by the susceptibletarget insect will result in reduced insect damage to the host plant,compared to plants which do not express the isp3 gene. A Lepidopteraninsect pest mainly damaging the roots can thus effectively be controlledby expressing an isp3 gene under a root specific promoter. A promoterpreferentially active in roots is described in WO00/29566. A suitablepromoter for root preferential expression is the ZRP promoter (andmodifications thereof) as described in U.S. Pat. No. 5,633,363. Anotheralternative is to use a promoter whose expression is inducible, forexample a wound-inducible promoter such as, e.g., the MPI promoterdescribed by Cordera et al. (1994), which is induced by wounding (suchas caused by insect feeding), or a promoter inducible by a chemical,such as dexamethasone as described by Aoyama and Chua (1997) or apromoter inducible by temperature, such as the heat shock promoterdescribed in U.S. Pat. No. 5,447,858, or a promoter inducible by otherexternal stimuli.

The insecticidally effective isp3 gene part may be inserted into theplant genome so that the inserted gene part is upstream (i.e., 5′) ofsuitable 3′ end transcription regulation signals (i.e., transcriptformation and polyadenylation signals). This is preferably accomplishedby inserting the isp3 chimeric gene in the plant cell genome. Suitablepolyadenylation and transcript formation signals include those of theCaMV 35S gene, the nopaline synthase gene (Depicker et al., 1982), theoctopine synthase gene (Gielen et al., 1984) and the T-DNA gene 7(Velten and Schell, 1985), which act as 3′-untranslated DNA sequences intransformed plant cells.

Introduction of the T-DNA vector into Agrobacterium can be carried outusing known methods, such as electroporation or triparental mating.

The insecticidally-effective isp3 gene part can optionally be insertedin the plant genome as a hybrid gene (U.S. Pat. NO. 5,254,799; Vaeck etal., 1987) under the control of the same promoter as a selectable orscorable marker gene, such as the neo gene (EP 0 242 236) encodingkanamycin resistance, so that the plant expresses a fusion protein thatis easily detectable.

Transformation of plant cells can also be used to produce the proteinsof the invention in large amounts in plant cell cultures, e.g., toproduce an ISP3 protein that can then be applied onto crops after properformulation. When reference to a transgenic plant cell is made herein,this refers to a plant cell (or also a plant protoplast) as such inisolation or in tissue culture, or to a plant cell (or protoplast)contained in a plant or in a differentiated organ or tissue, and bothpossibilities are specifically included herein. Hence, a reference to aplant cell in the description or claims is meant to refer not only toisolated cells in culture, but also to any plant cell, wherever it maybe located or in whatever type of plant tissue or organ it may bepresent.

All or part of the isp3 gene, encoding an anti-Lepidopteran protein, canalso be used to transform other microorganisms, including bacteria, suchas a B. thuringiensis, which has insecticidal activity againstLepidoptera or Coleoptera. Thereby, a transformed Bt strain can beproduced which is useful for combating a wide spectrum of Lepidopteranand/or Coleopteran insect pests or for combating additional Lepidopteraninsect pests. Transformation of bacteria, such as bacteria of the genusPseudomonas, Agrobacterium, Bacillus or Escherichia, with all or part ofthe isp3 gene of this invention, incorporated in a suitable cloningvehicle, can be carried out in a conventional manner, using, e.g.,conventional electroporation techniques as described in Mahillon et al.(1989) and in PCT Patent publication WO 90/06999.

Transformed Bacillus species strains containing the isp3 gene of thisinvention can be fermented by conventional methods (Dulmage, 1981;Bernhard and Utz, 1993) to provide high yields of cells. Underappropriate growth conditions, which are well understood, these strainssecrete ISP3 proteins in high yields.

Alternative suitable host microorganisms in which the isp3 genes can beexpressed are fungi, algae, or viruses, particularly species which areplant colonizing (e.g., (endo)symbiontic) species or insect pathogens.

An insecticidal, particularly anti-Lepidopteran, composition of thisinvention can be formulated in a conventional manner using themicroorganisms transformed with the isp3 gene, or their respective ISP3proteins or the ISP3 toxin, or an insecticidally effective toxin portionas an active ingredient, together with suitable carriers, diluents,emulsifiers and/or dispersants (e.g., as described by Bernhard and Utz,1993). This insecticide composition can be formulated as a wettablepowder, pellets, granules or dust or as a liquid formulation withaqueous or non-aqueous solvents as a foam, gel, suspension, concentrate,etc. Examples of compositions comprising insecticidal Bt spores aredescribed in WO96/10083.

A method for controlling insects, particularly Lepidoptera, inaccordance with this invention can comprise applying (e.g., spraying),to a locus (area) to be protected, an insecticidal amount of the ISP3proteins or compositions comprising the ISP3 proteins or comprising hostcells transformed with the isp3 genes of this invention. The locus to beprotected can include, for example, the habitat of the insect pests orgrowing vegetation (e.g. application to the foliage) or an area wherevegetation is to be grown (e.g. application to soil or water). In oneembodiment, a composition according to the present invention comprisesan insecticidal amount of at least one of the ISP3 proteins of theinvention, which may be produced by a bacterial host. Such a compositionmay be applied to leaves, soil, or seed coating.

The term “contacting” is used herein to mean, “to bring into physicalcontact with.” Contacting a plant with an insecticidal protein meansthat the insecticidal protein is brought into contact with cells of theplant, either internally (for example by expression in the plant) orexternally (for example by applying compositions comprising theinsecticidal protein externally to the plant). It is understood that theterm does not indicate the length of time of contact, but comprises anyperiod of contact (e.g. brief contact, long contact). When referring toa method of protecting a plant against insect damage comprisingcontacting said plant (or cells or tissues thereof) with an insecticidalprotein of the invention, the contact may be long enough and extensiveenough (with a high enough amount of protein contacting a large enoughnumber of cells) to prevent or reduce insect damage.

This invention further relates to a method for controlling Lepidopterancotton pests, such as bollworms, budworms, and earworms. SpecificLepidopteran cotton pests that may be controlled by the methods of thepresent invention include, but are not limited to, those selected fromthe group of Helicoverpa zea (Corn Earworm), Helicoverpa armigera(Cotton Bollworm), Helicoverpa punctigera (Native Bollworm), Heliothisvirescens (Tobacco Budworm), Spodoptera frugiperda (Fall Armyworm) andPectinophora gossypiella (Pink Bollworm). The method of controllingLepidopteran cotton pests comprises applying to an area or plant to beprotected, a ISP3 protein as defined herein, such as an ISP3-1099Eprotein and/or a ISP3-327D protein and/or ISP3-2245J protein, all asdefined herein. This may be accomplished by contacting a cotton plantwith an ISP3 protein of this invention, for example by planting a cottonplant transformed with an isp3 gene of this invention, or spraying acomposition containing a ISP3 protein of this invention. The inventionalso relates to the use of the ISP3 proteins of this invention,particularly the ISP3-1099E protein and/or ISP3-327D protein and/orISP3-2245J protein, against Lepidopteran cotton insect pests to minimizedamage to cotton plants.

This invention further relates to a method for controlling Lepidopteranmaize pests, such as earworms, armyworms, and corn borers. Specificmaize pests that may be controlled by the methods of the presentinvention may be selected from the group of Helicoverpa zea (CornEarworm), Agrotis ipsilon (Black Cutworm), Ostrinia nubilalis (EuropeanCorn Borer) and Spodoptera frugiperda (Fall Armyworm). The methodcomprises applying to an area or plant to be protected, a ISP3 proteinas defined herein, such as an ISP3-1099E protein and/or an ISP3-327Dprotein and/or an ISP3-2245) protein, all as defined herein. This may beaccomplished by contacting a maize plant with an ISP3 protein of thisinvention, for example by planting a maize plant transformed with anisp3 gene of this invention, or spraying a composition containing a ISP3protein of this invention. The invention also relates to the use of theISP3 proteins of this invention, such as the ISP3-1099E protein and/orISP3-327D protein and/or ISP3-2245J protein, against Lepidopteran maizeinsect pests to minimize damage to maize plants.

This invention further relates to a method for controlling Lepidopteranrice pests, such as rice stemborers, rice skippers, rice cutworms, ricearmyworms, rice caseworms, and rice leaffolders. Specific rice peststhat may be controlled by the methods of the present invention may beselected from the group of Yellow Stem Borer (Scirphophaga incertulas),Leaffolder (Cnaphalocrocis medinalis), Pink Stem Borer (Sesamiainferens) and Corn Spotted Stem Borer (Chilo partellus). The methodcomprises applying to an area or plant to be protected, a ISP3 proteinas defined herein, such as an ISP3-1099E protein and/or an ISP3-327Dprotein and/or an ISP3-2245J protein, all as defined herein. This may beaccomplished by contacting a rice plant with an ISP3 protein of thisinvention, for example by planting a rice plant transformed with an isp3gene of this invention, or spraying a composition containing a ISP3protein of this invention. The invention also relates to the use of theISP3 proteins of this invention, such as the ISP3-1099E protein and/orISP3-327D protein and/or ISP3-2245J protein, against Lepidopteran riceinsect pests to minimize damage to rice plants.

This invention further relates to a method for controlling Lepidopteransoybean pests. Specific soybean pests that may be controlled by themethods of the present invention may be selected from the group ofVelvet Bean Caterpillar (Anticarsia gemmatalis), Soybean Looper(Pseudoplusia includens), Beet Armyworm (Spodoptera exigua),Yellowstriped Armyworm (Spodoptera ornithogalli), Corn Earworm(Helicoverpa zea), Pod Borer (Epinotia aporema) and Rachiplusia nu. Thismethod comprises applying to an area or plant to be protected, a ISP3protein as defined herein, such as an ISP3-1099E protein and/or anISP3-327D protein and/or an ISP3-2245J protein, all as defined herein.This may be accomplished by contacting a soybean plant with an ISP3protein of this invention, for example by planting a soybean planttransformed with an isp3 gene of this invention, or spraying acomposition containing a ISP3 protein of this invention. The inventionalso relates to the use of the ISP3 proteins of this invention, such asthe ISP3-1099E protein and/or ISP3-327D protein and/or ISP3-2245Jprotein, against Lepidopteran soybean insect pests to minimize damage tosoybean plants.

To obtain the ISP3 toxin or protein, cells of the recombinant hostsexpressing the ISP3 protein can be grown in a conventional manner on asuitable culture medium. The secreted toxin can be separated andpurified from the growth medium. Alternatively, if the proteins are notsecreted, the cells can be lysed using conventional means such as enzymedegradation or detergents or the like. The toxin can then be separatedand purified by standard techniques such as chromatography, extraction,electrophoresis, or the like.

The term “gene” as used herein means any DNA or RNA fragment comprisinga region (the “transcribed region”) which may be transcribed into an RNAmolecule (e.g., an mRNA) in a cell, operably linked to suitableregulatory regions, e.g., a plant-expressible promoter. A gene may thuscomprise several operably linked fragments such as a promoter, a 5′leader sequence, a coding region, and a 3′ nontranslated sequence,comprising a polyadenylation site. A gene endogenous to a particularorganism (such as a plant species or a bacterial strain) is a gene,which is naturally found in that organism in nature. A “chimeric gene,”when referring to an isp3 DNA of this invention, refers to an isp3 DNAsequence having 5′ and/or 3′ regulatory sequences different from thenaturally-occurring bacterial 5′ and/or 3′ regulatory sequences, whichdrive the expression of the isp3 gene in its native host cell.

The term “expression of a gene” refers to the process wherein a DNA orRNA region which is operably linked to appropriate regulatory regions,such as to a promoter, is transcribed into an RNA which is biologicallyactive. A biologically active RNA is either capable of interaction withanother nucleic acid, or is capable of being translated into abiologically active polypeptide or protein. A gene is said to encode anRNA when the end product of the expression of the gene is biologicallyactive RNA, such as e.g. an antisense RNA, a ribozyme or a replicativeintermediate. A gene is said to encode a protein when the end product ofthe expression of the gene is a biologically active protein orpolypeptide

For the purpose of this invention the “sequence identity” of two relatednucleotide or amino acid sequences, expressed as a percentage, refers tothe number of positions in the two optimally aligned sequences whichhave identical residues (×100) divided by the number of positionscompared. A gap, i.e. a position in an alignment where a residue ispresent in one sequence but not in the other is regarded as a positionwith non-identical residues. To calculate sequence identity between twosequences for the purpose of this invention, the GAP program, which usesthe Needleman and Wunsch algorithm (1970) and which is provided by theWisconsin Package, Version 10.2, Genetics Computer Group (GCG), 575Science Drive, Madison, Wis. 53711, USA, may be used. The GAP parametersused are a gap creation penalty=50 (nucleotides)/8 (amino acids), a gapextension penalty=3 (nucleotides) /2 (amino acids), and a scoring matrix“nwsgapdna” (nucleotides) or “blosum62” (amino acids).

GAP uses the Needleman and Wunsch global alignment algorithm to aligntwo sequences over their entire length, maximizing the number of matchesand minimizes the number of gaps. The default parameters are a gapcreation penalty=50 (nucleotides)/8 (proteins) and gap extensionpenalty=3 (nucleotides)/2 (proteins). For nucleotides, the defaultscoring matrix used is “nwsgapdna” and for proteins the default scoringmatrix is “blosum62” (Henikoff & Henikoff, 1992).

The present invention includes, but is not limited to, the followingspecific embodiments.

An isolated insecticidal protein comprising an amino acid sequence withat least 91% sequence identity to SEQ ID NO: 4.

An isolated insecticidal protein comprising an amino acid sequence withat least 91% sequence identity to SEQ ID NO: 2.

An isolated insecticidal protein comprising an amino acid sequence withat least 88% sequence identity to SEQ ID NO: 6.

An isolated insecticidal protein comprising the amino acid sequence ofSEQ ID NO: 2 or the smallest toxic fragment thereof.

An isolated insecticidal protein comprising the amino acid sequence ofSEQ ID NO: 4 or the smallest toxic fragment thereof.

An isolated insecticidal protein comprising the amino acid sequence ofSEQ ID NO: 6 or the smallest toxic fragment thereof.

A protein according to any of paragraphs 109-114 above, wherein saidprotein is insecticidal against at least one insect species selectedfrom the group consisting of Helicoverpa zea, Heliothis virescens,Ostrinia nubilalis, Spodoptera frugiperda, Agrotis ipsilon, Pectinophoragossypiella, Scirphophaga incertulas, Cnaphalocrocis medinalis, Sesamiainferens, Chilo partellus and Anticarsia gemmatalis.

An isolated nucleic acid sequence encoding a protein according to anyone of paragraphs 109-114.

An isolated nucleic acid encoding a protein according to any one ofparagraphs 109-114, wherein said protein is insecticidal against atleast one insect species selected from the group consisting ofHelicoverpa zea, Heliothis virescens, Ostrinia nubilalis, Spodopterafrugiperda, Agrotis ipsilon, Pectinophora gossypiella, Scirphophagaincertulas, Cnaphalocrocis medinalis, Sesamia inferens, Chilo partellusand Anticarsia gemmatalis.

An isolated nucleic acid comprising a DNA sequence with at least 93%sequence identity to SEQ ID NO: 1.

An isolated nucleic acid comprising a DNA sequence with at least 94%sequence identity to SEQ ID NO: 3.

An isolated nucleic acid comprising a DNA sequence with at least 97%sequence identity to SEQ ID NO: 5.

An isolated nucleic acid comprising the nucleotide sequence fromnucleotide position 1 to nucleotide position 2364 in SEQ ID NO: 1.

An isolated nucleic acid comprising the nucleotide sequence fromnucleotide position 1 to nucleotide position 2367 in SEQ ID NO: 3.

An isolated nucleic acid comprising the nucleotide sequence fromnucleotide position 1 to nucleotide position 2361 in SEQ ID NO: 5.

An isolated nucleic acid comprising a nucleotide sequence which encodesan insecticidal protein comprising an amino acid sequence that is thetranslation product of a nucleic acid sequence which hybridizes to SEQID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 5 under stringent hybridizationconditions.

An isolated nucleic acid comprising a nucleotide sequence encoding aprotein according to any one of paragraphs 109-114 above, wherein saidnucleic acid has a synthetic nucleotide sequence that has been optimizedfor expression in monocotyledonous plants or dicotyledonous plants.

A chimeric gene comprising a promoter operably linked to a nucleic acidof any one of paragraphs 116-125 above.

A chimeric gene comprising a promoter operably linked to a nucleic acidcomprising a nucleotide sequence encoding the amino acid sequence of SEQID NO: 2 or the smallest toxic fragment thereof.

A chimeric gene comprising a promoter operably linked to a nucleic acidcomprising a nucleotide sequence encoding the amino acid sequence of SEQID NO: 4 or the smallest toxic fragment thereof.

A chimeric gene comprising a promoter operably linked to a nucleic acidcomprising a nucleotide sequence encoding the amino acid sequence of SEQID NO: 6 or the smallest toxic fragment thereof.

A vector comprising the chimeric gene of any one of paragraphs 126-129above.

A transgenic host cell comprising the chimeric gene of any one ofparagraphs 126-129 above.

A host cell according to paragraph 131, wherein said host cell is aplant cell.

A host cell according to paragraph 131, wherein said host cell is amicroorganism.

A transgenic plant comprising the chimeric gene of any one of paragraphs126-129, above.

A plant according to paragraph 134, wherein said plant is a maize,cotton, rice or soybean plant.

A method of protecting a plant against insect damage comprisingcontacting said plant with an insecticidal protein, wherein said proteincomprises the amino acid sequence of SEQ ID NO: 2.

A method of protecting a plant against insect damage comprisingcontacting said plant with an insecticidal protein, wherein said proteincomprises the amino acid sequence of SEQ ID NO: 4.

A method of protecting a plant against insect damage comprisingcontacting said plant with a insecticidal protein, wherein said proteincomprises the amino acid sequence of SEQ ID NO: 6

A method according to any one of paragraphs 136-138 above, wherein saidinsecticidal protein is encoded by a chimeric gene integrated in thegenome of said plant.

A method according to any one of paragraphs 136-138 above, wherein saidprotein is applied externally to said plant.

A method according to any one of paragraphs 136-138 above, wherein saidplant is a maize, cotton, soybean or rice plant.

An isolated Bacillus thuringiensis strain comprising a nucleic acidsequence according to paragraph 116, above.

An insecticidal composition comprising a protein of any one ofparagraphs 109-114 above, which, when applied externally to a plant,increases resistance to insect damage compared to control plants towhich such composition is not applied.

The method of evolving a nucleic acid sequence encoding an ISP3 proteincomprises the following steps:

-   a) providing a population of nucleic acid sequences encoding the    amino acid sequences of SEQ ID NO: 2 and/or SEQ ID NO: 4 and/or SEQ    ID NO: 6, or variants or fragments of the amino acid sequences of    SEQ ID NO: 2 and/or SEQ ID NO: 4 and/or SEQ ID NO: 6, wherein said    variants or fragments have a sequence identity of at least 91% to    SEQ ID NO: 2 or SEQ ID NO: 4 and at least 88% to SEQ ID NO: 6;-   b) shuffling said population of variants or fragments to form    recombinant nucleic acid molecules;-   c) selecting or screening for recombinant nucleic acid molecules,    which encode proteins that have insecticidal activity; and-   d) repeating steps (a) to (c) with the recombinant nucleic acid    molecules selected in step (c) until a recombinant nucleic acid    molecule has been found in step (c), wherein the protein encoded by    said nucleic acid molecule has the desired insecticidal property.

These and/or other embodiments of this invention are reflected in theclaims, which form part of the description of the invention.

The following Examples illustrate the invention, and are not provided tolimit the invention or the protection sought. The sequence listingreferred to in the Examples, the Claims and the Description is asfollows:

-   SEQ ID NO: 1: DNA sequence isp3-1099E-   SEQ ID NO: 2: amino acid sequence ISP3-1099E-   SEQ ID NO: 3: DNA sequence isp3-327D-   SEQ ID NO: 4: amino acid sequence ISP3-327D-   SEQ ID NO: 5: DNA sequence of isp3-2245J-   SEQ ID NO: 6: amino acid sequence of ISP3-2245J.

Unless stated otherwise in the Examples, all recombinant DNA techniquesare carried out according to standard protocols as described in Sambrookand Russell (2001) Molecular Cloning: A Laboratory Manual, ThirdEdition, Cold Spring Harbor Laboratory Press, NY, in Volumes 1 and 2 ofAusubel et al. (1994) Current Protocols in Molecular Biology, CurrentProtocols, USA and in Volumes I and II of Brown (1998) Molecular BiologyLabFax, Second Edition, Academic Press (UK). Standard materials andmethods for plant molecular work are described in Plant MolecularBiology Labfax (1993) by R. D. D. Croy, jointly published by BIOSScientific Publications Ltd (UK) and Blackwell Scientific Publications,UK. Standard materials and methods for polymerase chain reactions can befound in Dieffenbach and Dveksler (1995) PCR Primer: A LaboratoryManual, Cold Spring Harbor Laboratory Press, and in McPherson at al.(2000) PCR—Basics: From Background to Bench, First Edition, SpringerVerlag, Germany.

It should be understood that the preceding is merely a detaileddescription of particular embodiments of this invention and thatnumerous changes to the disclosed embodiments can be made in accordancewith the disclosure herein without departing from the spirit or scope ofthe invention. The preceding description, therefore, is not meant tolimit the scope of the invention. Rather, the scope of the invention isto be determined only by the appended claims and their equivalents.

EXAMPLES Example 1 Characterization of Bacterial Strains

A bacterial strain, named herein BTS01099E, was isolated from grain dustfrom Belgium. A further bacterial strain, named herein BTS00327D, wasisolated from horse dung from Spain. A further bacterial strain, hereinnamed BTS02245J, was isolated from grain dust from the Philippines.

Each strain was grown overnight on LB agar plates (LB medium with 1.5%agar added; LB medium:10 g/l trypton, 10 g/l NaCl, 5 g/l yeast extract,pH 7.3) at 28° C. For small-scale cultures, 20 ml TB medium (TerrificBroth: 12 g/l tryptone, 24 g/l yeast extract, 3.8 g/l KH₂PO₄, 12.5 g/lK₂HPO₄, 5 ml/l glycerol, pH 7.1) was inoculated and grown for 65 hoursat 28° C. on a rotating platform having about 70 rotations per minute.After 65 hours, a protease inhibitor mixture was added to the culture.This cocktail has the following ingredients (volumes given are thoserequired to add to one 20 ml culture): 200 μl PMSF (100 mM), 200 μl of amixture of benzamidine HCl (100 mM) and epsilon-amino-n-caproic acid(500 mM), 400 μl EGTA (0.5M), 40 μl antipain (0.5 mg/ml)/leupeptin (0.5mg/ml) and 20 μl beta-mercapto ethanol (14M).

The culture medium to which the protease inhibitor mixture had beenadded was then centrifuged for 20 minutes at 3000 rpm. In some cases,the supernatant was concentrated about 4 times using Centriprep YM-10Centrifugal Filter Devices (Millipore Cat. No. 4305).

For long term storage, a loop of sporulated cells was added to 0.5 ml of25% glycerol and after vortexing, stored at −70° C. Sporulated cellswere obtained by growth of the strain on LB agar plates untilsporulation (as visible under the light microscope).

After cultivating on LB agar plates of single cell colonies, microscopicanalysis of the strain cultures of BTS01099E, BTS00327D and BTS02245Jshowed the presence of rod-shaped, motile, single, vegetative cells andsporangia containing an oval spore. Parasporal crystals were detected incultures of BTS00327D, BTS01099E and BTS02245J. Based on the rod-likeshape, the aerobic growth, and the presence of parasporal crystals,these three strains are believed to be B. thuringiensis species strains.

Each strain can be cultivated on conventional standard media, preferablyT₃ medium (tryptone 3 g/l, tryptose 2 g/l, yeast extract 1.5 g/l, 5 mgMnCl₂, 0.05 M Na₂HPO₄.2H₂O, 0.05 M NaH₂PO₄.H₂O, pH 6.8 and 1.5% agar),preferably at 28° C. For long-term storage, an equal volume of aspore-crystal suspension may be mixed with an equal volume of 50%glycerol and store this at −70° C. or lyophilize a spore-crystalsuspension. For sporulation, growth on T₃ medium is preferred for 72hours at 28° C.

Example 2 Insect Bioassays of Bacillus Strains (Using CultureSupernatant Containing Insecticidal Protein).

Bioassays were performed on neonate larvae of Helicoverpa zea, Heliothisvirescens, Ostrinia nubilalis, Spodoptera frugiperda and Agrotisipsilon. Cell-free supernatant of bacterial cultures of differentBacillus strains was used in insect bioassays (“surface contaminationassay”) against various lepidopteran insects.

Strain BTS01099E, BTS00327D or BTS02245J was grown at 28° C. in TBmedium. Cell-free culture supernatant was harvested 65 hours afterculture initiation, dilutions were made and applied onto the surface ofsolidified artificial diet. This diet, based on Vanderzand 1962,comprises: agar 20 g, water 1000 ml, corn flour 96 g, yeast 30 g, wheatgerm 64 g, Wesson salt 7.5 g, casein 15 g, sorbic acid 2 g, Aureomycin0.3 g, Nipagin 1 g, wheat germ oil 4 ml, sucrose 15 g, cholesterol 1 g,ascorbic acid 3.5 g, and Vanderzand mod. vit. mix 12 g. The diet wasdispensed in wells of Costar 48-well plates and allowed to solidify. 25μl supernatant solution was applied onto the surface of each well (1cm²). One neonate (L1; first instar) insect larva was placed on eachwell, and 18-20 larvae were used per sample. Dishes were kept at 25±1°C. and mortality rates (percentage dead versus living larvae) wererecorded after 7 days. As a negative control standard diet and TB wasused.

Results (shown in Table 1 below) showed that the cell-free supernatantsof strains BTS01099E, BTS00327D and BTS02245J showed toxicity towardsHeliothis virescens, Helicoverpa zea and Ostrinia nubilalis larvae. Inaddition, the supernatant of strain BTS02245J also showed toxicitytowards Spodoptera frugiperda larvae and the supernatant of BTS00327Dshowed toxicity towards Agrotis ipsilon larvae.

TABLE 1 Hz mort On mort Sf mort Ai mort Strain Hv mort (%) (%) (%) (%)(%) BTS02245J 11 (ir)/33 (gi) 94 (gi)/50  50 (gi) 78 (gi) nt BTS00327D70 35-78 (gi) 100 nt 100 BTS01099E 25 10  46 nt nt Hv: Heliothisvirescens, Hz: Helicoverpa zea; On: Ostrinia nubilalis, Sf: Spodopterafrugiperda; Ai: Agrotis ipsilon; nt: not tested

Negative controls (standard diet):

-   BTS02245J—Hv 9%, Hz 0%, On 0%, Sf 0%-   BTS00327D—Hv 10%, Hz 6%, On 4%, Ai 0%-   BTS01099E—Hv 10%, Hz 0%, On 0%

Additional Observations:

gi: growth inhibition of larvae (live larvae still in L1/L2 instar stageafter 7 days); ir: irregular growth of larvae (a proportion of livelarvae in L1/L2 instar stage, a proportion of live larvae in L3/L4instar stage after 7 days)

Cell-free supernatant of strain BTS02245J caused 11% and 33% mortalityof H. virescens larvae, 94% and 50% mortality of H. zea larvae, 50%mortality of O. nubilalis larvae and 78% mortality of S. frugiperdalarvae, showing that supernatant of this strain has insecticidalactivity, particularly against H. zea and S. frugiperda. Toxicity islikely caused by a protein secreted by this strain into the culturemedium.

The cell-free supernatant of strain BTS00327D caused 70% mortality of H.virescens larvae, 35% to 78% mortality of H. zea larvae, 100% mortalityof O. nubilalis larvae and 100% mortality of Agrotis ipsilon larvae.Thus, the supernatant of this strain showed strong toxicity to at leastfour different species of Lepidopteran insects. Toxicity is likely to becaused by an insecticidally active protein secreted by this strain intothe culture medium.

The cell-free supernatant of strain BTS01099E caused 25% mortality of H.virescens larvae, 10% mortality of H. zea larvae and 46% mortality of O.nubilalis larvae, indicating that the supernatant of this strain hastoxic activity against different species of Lepidopteran insects.Toxicity is likely to be caused by an insecticidally active proteinsecreted by this strain into the culture medium.

Example 3 Cloning of isp3 Genes

Cloning of the nucleotide sequence encoding ISP3-1099E from strainBTS01099E.

Total DNA of strain BTS01099E was prepared and partially digested withSau3A. The digested DNA was size fractioned on a sucrose gradient.Fragments ranging from 7 kb to 10 kb were ligated to cloning vectorpUC19I (a derivative of pUC19; Yannisch-Perron et al. 1985), after BamH1digestion and treatment of the cloning vector with TsAP (thermosensitivealkaline phosphatase). The ligation mixture was then electroporated intoE. coli XL1-Blue cells. Transformants were plated on LB-triacillinplates containing X-gal and IPTG and white colonies were selected foruse in filter hybridization experiments. Recombinant E. coli clonescontaining the vector were then screened with a DIG labeled probe, whichwas prepared as follows. First, a PCR was performed using as templatecells from strain BTS01099E. The resulting amplification product wasgel-purified and cloned into pGEM-T. The resulting plasmid was used astemplate in a PCR reaction using DIG-labeled dNTPs and the same primersas in the first PCR reaction. An appropriate amount of thisamplification product was used in hybridization reactions.

Following the identification of a positive colony containing a plasmidharboring the full-length isp3 gene, the sequence of this gene wasdetermined using the dye terminator labeling method and a Perkin ElmerABI Prism-377 DNA sequencer. Both the coding and non-coding strands weresequenced.

The sequence of the open reading frame found in the cloned DNA fragmentof a positive colony is shown in SEQ ID NO: 1 (isp3-1099E). This DNAsequence was found to encode the novel protein shown in SEQ ID NO: 2(ISP3-1099E).

To show that this DNA sequence is the cause of the insecticidal activityobserved, the sequence was expressed in a bacterial strain and thesupernatant or cell lysate of the recombinant strain was tested forinsecticidal activity in insect bioassays.

Cloning of the nucleotide sequence encoding ISP3-327D from strainBTS00327D

Total DNA of strain BTS00327D was prepared and partially digested withSau3A. The digested DNA was size fractioned on a sucrose gradient.Fragments ranging from 7 kb to 10 kb were ligated to cloning vectorpUC19I (a derivative of pUC19; Yannisch-Perron et al. 1985), after BamH1digestion and treatment of the cloning vector with TsAP (thermosensitivealkaline phosphatase). The ligation mixture was then electroporated intoE. coli XL1-Blue cells. Transformants were plated on LB-triacillinplates containing X-gal and IPTG and white colonies were used in filterhybridization experiments. Recombinant E. coli clones containing thevector were then screened with a DIG labeled probe, which was preparedas follows. First, a PCR was performed using as template cells fromstrain BTS00327D. The resulting amplification product was gel-purifiedand cloned into pGEM-T. The resulting plasmid was used as template in aPCR reaction using DIG-labeled dNTPs and the same primers as in thefirst PCR reaction. An appropriate amount of this amplification productwas used in hybridization reactions.

Following the identification of a positive colony containing a plasmidharboring the full-length isp3 gene, the sequence of this gene wasdetermined using the dye terminator labeling method and a Perkin ElmerABI Prism-377 DNA sequencer. Both the coding and non-coding strands weresequenced.

The sequence of the open reading frame found in the cloned DNA fragmentof a positive colony is shown in SEQ ID NO: 3 (isp3-327D). This DNAsequence was found to encode the novel protein shown in SEQ ID NO: 4(ISP3-327D).

To show that this DNA sequence is the cause of the insecticidal activityobserved, the sequence was expressed in a bacterial strain and thesupernatant or cell lysate of the recombinant strain was tested forinsecticidal activity in insect bioassays.

Cloning of the nucleotide sequence encoding ISP3-2245J from strainBTS02245J

Total DNA of strain BTS02245J was prepared and partially digested withSau3A. The digested DNA was size fractioned on a sucrose gradient.Fragments ranging from 7kb to 10kb were ligated to cloning vector pUC19I(a derivative of pUC19; Yannisch-Perron et al. 1985), after BamH1digestion and treatment of the cloning vector with TsAP (thermosensitivealkaline phosphatase). The ligation mixture was then electroporated intoE. coli XL1-Blue cells. Transformants were plated on LB-triacillinplates containing X-gal and IPTG and white colonies were used in filterhybridization experiments. Recombinant E. coli clones containing thevector were then screened with a DIG labeled probe, which was preparedas follows. First, a PCR was performed using as template cells fromstrain BTS02245J. The resulting amplification product was gel-purifiedand cloned into pGEM-T. The resulting plasmid was used as template in aPCR reaction using DIG-labeled dNTPs and the same primers as in thefirst PCR reaction. An appropriate amount of this amplification productwas used in hybridization reactions.

Following the identification of a positive colony containing a plasmidharboring the full-length isp3 gene, the sequence of this gene wasdetermined using the dye terminator labeling method and a Perkin ElmerABI Prism-377 DNA sequencer. Both the coding and non-coding strands weresequenced.

The sequence of the open reading frame found in the cloned DNA fragmentof a positive colony is shown in SEQ ID NO: 5 (isp3-2245J). This DNAsequence was found to encode the novel protein shown in SEQ ID NO: 6(ISP3-2245J).

To show that this DNA sequence is the cause of the insecticidal activityobserved, the sequence was expressed in a bacterial strain and thesupernatant or cell lysate of the recombinant strain was tested forinsecticidal activity in insect bioassays.

Example 4 Recombinant Expression of ISP3 Proteins in E. coli

SEQ ID NO: 1 (isp3-1099E), SEQ ID NO: 3 (isp3-327D) and SEQ ID NO: 5(isp3-2245J) were subcloned into an expression vector, under control ofthe cry1Ab promoter, and expressed in E. coli strain WK6. Duringsubcloning, an NcoI restriction site was introduced at the ATG startcodon, thereby changing the second amino acid of SEQ ID No 2, SEQ ID No4 and SEQ ID NO: 6 from Asparagine (Asn) into Aspartate (Asp).

SDS-PAGE analysis of transformed E. coli cell lysates showed thatproteins of the expected molecular weight (±88 kDa) were produced foreach of the three genes. As negative controls cell lysate ofnon-transformed E. coli WK6 were used.

Cell lysate of recombinant E. coli cultures, expressing isp3-327D andisp3-1099E, was used in insect bioassays (surface contamination assays)as described in Example 5. The results are summarized in Table 2 below.Plus symbols indicate significant insect mortality over the negativecontrol.

TABLE 2 Gene in E. coli Hz Hv Sf Ag Dvv isp3-327D + + + + −isp3-1099E + + + + − negative control WK6 − − − − − Hz: Helicoverpa zea,Hz: Heliothis virescens, Sf: Spodoptera frugiperda, Dvv: Diabroticavirgifera virgifera, Ag: Anticarsia gemmatalis

Bioassays:

Hz: Surface contamination on heliothis food in 48 multiwell Costarplates, 25 μl/well (1 cm²), 18×1 L1 per concentration

Hv: Surface contamination on heliothis food in 24 multiwell Costarplates, 50 μl/well (2 cm²), 20×1 L1 per concentration

Sf: surface contamination on littoralis food in 48 multiwell Costarplates; 25 μl/well (1 cm²); 18×1 L1 per concentration

Ag: surface contamination on littoralis food in 24 multiwell Costarplates, 50 μl/well (2 cm²), 12×2 L1 per concentration

Incubation at T:25±1° C.; Score after 7 days.

For ISP3-327D protein and ISP3-1099E protein, significant mortality wasfound in surface contamination assays with Helicoverpa zea, Heliothisvirescens, Spodoptera frugiperda and Anticarsia gemmatalis. In addition,undiluted cell lysate of recombinant E. coli expressing ISP3-327D orISP3-1099E showed significant mortality against Ostrinia nubilalis (50%(gi) mortality for ISP3-327D, 26% (gi) mortality for ISP3-1099E comparedto 0% mortality for the control WK6).

Example 5 Recombinant Expression of ISP3 Proteins in Bt

SEQ ID NO: 1 (isp3-1099E) and SEQ ID NO: 5 were subcloned into a shuttlevector and expressed in a crystal minus strain Bt strain (IPS 78/11 orBerliner 1715cry⁻). In bioassays, supernatant from the non-transformedBt strain is used as negative control.

The cell-free culture supernatant from the recombinant Bt strain istested for toxicity against Lepidopteran insect larvae, such as H.virescens, H. zea, H. armigera, O. nubilalis, S. frugiperda, Agrotisipsflon, Pectinophora gossypiella and A. gemmatalis, using a surfacecontamination assay as described above (Example 2) and below (todetermine LC₅₀ values).

Supernatant from the recombinant Bt strain is obtained as follows: TheBt strain is grown overnight on LB agar plates containing erythromycin(20 μg/ml) at 28° C. For small-scale cultures, 20 ml TB mediumcontaining erythromycin (20 μg/ml) is inoculated and grown for 65 hoursat 28° C. on a rotating platform having about 70 rotations per minute.After 65 hours, a protease inhibitor mixture is added to the culture.This cocktail has the following ingredients (volumes given are thoserequired to add to one 20 ml culture): 200 μl PMSF (100 mM), 200 μl of amixture of benzamidine HCl (100 mM)/epsilon-amino-n-caproic acid (500mM), 40011 EGTA (0.5M), 40 μl antipain (0.5 mg/ml)/leupeptin (0.5 mg/ml)and 20 μl beta-mercaptoethanol (14M). The culture medium, to which theprotease inhibitor mixture had been added, is then centrifuged for 20minutes at 3000 rpm. In some cases, the supernatant is concentratedabout 4 times using centriprep YM-10 Centrifugal Filter Devices(Millipore, Cat. No. 4305).

For Helicoverpa zea and Heliothis virescens the following artificialdiet is used in the surface contamination assay: water 1 liter, agar 20g, soyflour 81 g, wheat germ 36 g, Wesson salt mix 10 g, sucrose 14.7 g,Nipagin 1 g, sorbic acid 1.1 g, Vanderzant vit.mix. 9.5 g, corn oil 5ml, Nystatin 0.06 g, and Aureomycin 0.17 g.

In the surface contamination assays for other lepidopteran insects, thefollowing artificial diet is used: water 1 liter, agar, 20 g, corn flour112 g, wheatgerm 28 g, yeast 30 g, sorbic acid 1.2 g, Nipagin 1 g,Aureomycin 0.06 g, Nystatin 0.03 g, and ascorbic acid 4.8 g.

The artificial diet is dispensed in wells of Costar 24-multiwell platesand allowed to solidify. 50 μl of diluted supernatant is applied ontothe surface of each well (2 cm²). One or two neonate (L1; first instar)larvae are placed on each well (depending on the species, e.g. for O.nubilalis 2 larvae/well) and around 20 to 24 larvae are used persupernatant dilution. Six to eight supernatant dilutions (the dilutionfactor is around 3), ranging from about 4050 to 0.21 ng/cm² are tested.Dishes are kept at 25±1° C. and mortality rates (percentage dead versusliving larvae) are recorded after 7 days. As a negative control standarddiet and TB is used. LC₅₀ values and/or LC₉₀ values are calculated withprobit analysis using the program POLO PC (from LeOra Software, 1987,Berkely, Calif.). The LC₅₀ value is the total supernatant proteinconcentration when 50% of the tested insect larvae are killed.

The bioassays show that the proteins encoded by the cloned sequencesisp3-1099E and isp3-2245J each cause significant insecticidal activityagainst selected Lepidopteran insects.

SEQ ID NO: 3 (isp3-327D) was subcloned into a shuttle vector andexpressed in the crystal minus Bt strain IPS78/11. In bioassays,supernatant from the non-transformed Bt strain was used as negativecontrol.

SDS-PAGE analysis showed that the culture supernatant contained aprotein with a molecular weight close to the calculated molecular weightof the ISP3-327D protein (±88 kDa).

Using the surface contamination assay as described in Example 2, thecell-free culture supernatant of Bt strain IPS78/11 expressing SEQ IDNO: 3 (isp3-327D) showed significant insecticidal activity againstOstrinia nubilalis (On), Pectinophora gossypiella (Pg) and Helicoverpazea (Hz), as shown in Table 3.

TABLE 3 Hz mort (%) On mort (%) Pg mort (%) Dvv mort (%) isp3-327D 94(gi)/58 (gi) 19 (gi) 29 (gi) 0 in IPS 78/11 Control  0  6  0 5 IPS 78/11gi: growth inhibition of larvae/Dvv: Diabrotica virgifera virgifera

Bioassays:

Surface contamination assays, as described above, using concentratedsupernatant after 26 hrs culture in TB and following addition ofprotease inhibitor cocktail. Heliothis artificial diet (as above) wasused for H. zea and littoralis artificial diet for O. nubialis and P.gossypiella. For H. zea and O. nubialis 48-well Costar plates, 25μl/well (1 cm²), 18 wells with one L1 larva per well, were used. For P.gossypiella 24-well Costar plates, 50 μl/well (2 cm²), and 12 wells withtwo L1 larvae per well were used. Dvv artificial diet (as above) wasused for Dvv in 24 well plates, 50 μl/well (2 cm²), and 12 wells withtwo larvae per well were used. Dvv artificial diet (as above) was usedfor Dvv in 24-well plates, 50 μl/well (2 cm²), 6 wells with 4 L1/well.

The bioassay showed that the protein encoded by the cloned sequenceisp3-327D has significant insecticidal activity against selectedLepidopteran insects.

Example 6 Further Characterization of ISP3-327D

Supernatant from the crystal-minus Bt strain IPS78/11 expressing SEQ IDNO: 3 (isp3-327D) was used to test trypsin digestability of ISP3-327Dprotein and toxicity of the resulting fragments. Trypsin treatment ofsupernatant of the transformed IPS78/11 culture resulted in two majorbands of about 65 kDa and about 23 kDa, as determined by SDS-PAGEanalysis.

Both trypsin-treated supernatant (4 hours treatment; reaction wasstopped with PMSF of a final concentration of 0.1 mM) andnon-trypsin-treated supernatant were used in surface contaminationassays against Helicoverpa zea. The surface contamination assay wasperformed on heliothis food in 48 multiwell plates (25 μl/well; 1 cm²).Six supernatant dilutions were tested. Per dilution, 18 wells and one L1larva per well were used. Mortality was scored after incubation at25°±1° C. after 7 days.

LC50 values for the untreated and trypsin-treated ISP3-327D proteinshowed overlapping 90% confidence intervals, showing that thetrypsin-treated protein retains toxic activity against H. zea.

Example 7 Rice Insect Bioassays of Recombinant ISP3 Proteins

The sequences for isp3-1099E (SEQ ID NO: 1), isp3-327D (SEQ ID NO: 3)and isp3-2245J (SEQ ID NO: 5) were expressed in E. coli as describedabove.

Cell lysates of recombinant E. coli were tested in insect bioassays foractivity against four Lepidopteran rice pests. Five to six doses perprotein were tested.

(a) Yellow Stem Borer (Scirphophaga incertulas) Bioassay:

Yellow Stem Borer adults were collected from rice fields. Eggs laid bythe adults were collected and kept in Petri dishes for hatching at 30°C. The newly hatched larvae were used in the bioassays.

Rice stalk sheaths of 6 cm were used. Six-centimeter-long segments ofthe leaf sheaths were cut from freshly excised rice stalks of thesusceptible variety TN1. The innermost part of the segment, along withone sheath cover, was separately dipped into the different protein dosesfor 30 seconds. The treated stalk sheath was immobilized vertically on 2cm agar gel in specimen tubes (7 cm long; 2.5 cm diameter). Ten to 15neonate larvae of Yellow Stem Borer were added to each treated stalk andtubes were sealed and incubated for five days. After 5 days, the numbersof surviving and of dead larvae were counted.

(b) Leaffolder (Cnaphalocrocis medinalis) Bioassay:

Insects were collected from rice fields in northern India. Insect larvaewere reared on rice plants in the green house. Emerging adults wereconfined in an oviposition chamber and oviposited plants were kept inwater trays for larval hatching. One-day-old larvae were used inbioassays.

Bioassays were conducted in cylindrical chambers, using freshly excisedleaves from rice plants at tillering stage. An 8 cm long leaf lamina wasexcised from the central whorl of the rice tiller. The leaf lamina wasplaced in the chamber and the different protein dosages applied. After30 minutes, five to ten larvae were added per leaf. At least threeleaves were used per protein dose. Five days after incubation, thenumber of dead and surviving larvae was counted.

(c) Pink Stem Borer (Sesamia inferens) Bioassay:

Insects were collected from rice fields in northern India and reared onrice plants.

Bioassays were conducted in glass vials (7.5 cm×2.5 cm diameter) usingan artificial diet made of dry bean powder, brewers yeast, sorbic acid,ascorbic acid, methyl paraben, agar and water. The diet was dispensedinto the vials up to 2 cm depth. At least three vials were prepared perprotein dose. 40 μl of test dose was spread uniformly onto the surfaceof the diet in each vial and left to dry for one hour. Ten to fifteenfirst instar larvae of Pink Stem Borer were added per vial. After sevendays, the number of dead and surviving larvae was counted.

(d) Corn Spotted Stem Borer (Chilo partellus) Bioassays:

Insects were maintained on artificial diet made up of red bean powder,brewers yeast, sorbic acid, sorghum leaf powder, ascorbic acid, methylpara-hydroxy benzoic acid, vitamins, wheat germ oil, Wesson saltmixture, agar, formaldehyde and water. Neonate larvae from thesecultures were used in the bioassays. The bioassays were performed asdescribed for Pink Stem Borer (Sesamia inferens).

e) Results of Rice Insect Bioassays

The results of the insect bioassays, summarized in table 4 below, showedthat ISP3-327D and ISP3-1099E were highly toxic to four Lepidopteranrice pests, namely Yellow Stem Borer (Scirphophaga incertulas),Leaffolder (Cnaphalocrocis medinalis), Pink Stem Borer (Sesamiainferens) and Corn Spotted Stem Borer (Chilo partellus). Further,ISP3-2245J protein showed significant toxicity towards threelepidopteran rice pests, namely Yellow Stem Borer (Scirphophagaincertulas), Leaffolder (Cnaphalocrocis medinalis) and Pink Stem Borer(Sesamia inferens), while no toxicity of ISP3-2245J protein against CornSpotted Stem Borer (Chilo partellus) was detected.

TABLE 4 Gene in E. coli YSB LF PSB SSB isp3-327D + + + +isp3-1099E + + + + Isp3-2245J + + + − negative control − − − − YSB:Yellow Stem Borer, LF: Leaffolder, PSB: Pink Stem Borer, SSB: SpottedStem Borer; plus symbols (+) indicate significant mortality over thenegative control.

Example 8 Production of ISP3 Proteins in Transformed Plants

Plant expression vectors are constructed comprising a plant-expressiblepromoter, operably linked to a DNA sequence encoding ISP3-1099E,ISP3-327D or ISP3-2245J (or a toxic fragment or variant thereof), and a3′ polyadenylation signal. A leader sequence, such as that from thechlorophyll a/b binding protein gene from Petunia (Harpster et al.1988), is inserted 5′ of the isp3 DNA. Codon usage of isp3-1099E,isp3-327D and isp3-2245J may be adapted to that of the host plant (e.g.corn, cotton or rice), for example as described in WO4/24264.

The promoters used to drive the isp3 genes are selected from theconstitutive promoters CaMV 35S (Hull and Howell, 1987), maize ubiquitinpromoter, rice actin promoter and Cassava Vein Mosaic Virus promoter. As3′ transcript termination and polyadenylation signal the 3′ 35S, 3′nos(Depicker et al. 1982), 3′ocs or 3′gene7 are used. For Agrobacteriummediated transformation, the expression cassette is inserted into aT-DNA vector, between the right and left border sequence.

Transformation of corn (Zea mays), cotton (Gossypium hirsutum orGossypium barbadense) and rice (Oryza sativa) plants.

Corn cells are stably transformed by Agrobacterium-mediatedtransformation as described in U.S. Pat. No. 6,140,553, incorporatedherein by reference. Cotton cells are stably transformed byAgrobacterium mediated transformation as described in WO 00/71733,incorporated herein by reference. Rice cells are stably transformed asdescribed in WO92/09696.

Transformed cells and plantlets are regenerated as described in theabove references. For constructs additionally comprising a selectablemarker gene, such as a herbicide resistance gene, for example 2mEPSPS(EPO 508 909 and EP 0 507 698 incorporated by reference) conferringresistance to glyphosate or the bar or PAT gene conferring resistance toglufosinate ammonium (EP 0 242 236 and EP 0 242 246 incorporated byreference) transformed cells are grown on selection media containing theselective agent, so that most of the selected regenerants aretransformed.

From the regenerants, transformants expressing the isp3 gene areselected by ELISA, Southern blot, Northern blot and/or PCR analysis.Transformation events, particularly single copy events, are thenselected for insecticidal activity towards Lepidopteran insect pests(using bioassays). Chimeric isp3-1099E, isp3-327D or isp3-2245)gene-containing progeny plants show improved resistance to lepidopteraninsects compared to non-transformed control plants. Particularly plantswith high levels of insect tolerance express a high level of ISP3protein and isp3 mRNA.

Transformants are further selected for agronomic characteristics (normalphenotype, yield, plant architecture, etc.). Following seed increases,field trials of transformed corn, cotton or rice plants are carried outin different regions where susceptible insect pests are present,demonstrating that plants expressing ISP3 proteins have an increasedinsect tolerance under field conditions in different environments.Following infestation (artificial or natural) of the field by insectpests, yield losses of the transformed plants are reduced compared tonon-transformed control plants.

To generate plants with broad insecticidal activity, the ISP3 expressingevents are crossed to each other and to other transgenic plants,expressing insecticidal proteins with a different (e.g., complementary)insecticidal spectrum. Progeny of the crosses are assayed forinsecticidal activity using bioassays for a range of different insectpests. Plants with insecticidal activity against the desired insectrange are generated in this way.

This invention is not limited to the above corn, cotton, soybean or riceplants, transformation methods, vector constructs (promoters, 3′ends,etc.) or the particular ISP3 proteins or DNA sequences used. Theinvention includes variants or equivalents of the ISP3 proteinsretaining insecticidal activity.

REFERENCES

An et al. (1996) Plant J. 10, 107.

Aoyama and Chua (1997) Plant Journal 11:605-612

Ausubel et al. (1994) Current Protocols in Molecular Biology, CurrentProtocols, Vol. 1 and 2, USA.

Bennetzen & Hall (1982) J. Biol. Chem. 257, 3026-3031.

Bernhard, K. and Utz, R. (1993) “Production of Bacillus thuringiensisinsecticides for experimental and commercial uses”, In Bacillusthuringiensis, An Environmental Biopesticide: Theory and Practice, pp.255-267, eds. Entwistle, P. F., Cory, J. S., Bailey, M. J. and Higgs,S., John Wiley and Sons, New York.

Bih et al. (1999), J. Biol. Chem. 274, 22884-22894.

Brown (1998) Molecular Biology LabFax, Volumes I and II, Second Edition,Academic Press (UK)

Callis et. al. (1987) Genes Developm. 1:1183-1200

Christensen et al. (1992) Plant Mol. Biol. 18, 675-689.

Cordera et al. (1994) The Plant Journal 6, 141.

Cornejo et al. (1993) Plant Mol. Biol. 23, 567-581.

Cornelissen et al. (1986) EMBO J. 5, 37-40.

Datta et al. (1990) Bio/Technology 8, 736-740

De Pater et al. (1992) Plant J. 2, 834-844.

Depicker et al. (1982) J. Molec. Appl. Genetics 1, 561-573.

Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, ColdSpring Harbor Laboratory Press

Doss et al (2002) Protein Expression and Purification 26, 82-88

Dulmage, H. T. (1981) “Production of Bacteria for Biological Control ofInsects” in Biological Control in Crop Production, Ed. Paparizas, D. C.,Osmun Publishers, Totowa, N.J., USA, pp. 129-141.

Estruch et al. (1996) Proc Natl Acad Sci USA 93, 5389-94.

Franck et al. (1980) Cell 21, 285-294

French et al. (1986) Anal. Biochem. 156, 417-423

Ffrench-Constant and Bowen (2000) Cell Mol Life Sci 57, 828-33.

Fromm et al. (1990) Bio/Technology 8, 833-839

Gardner et al. (1981) Nucleic Acids Research 9, 2871-2887

Ge et al. (1991) J. Biol. Chem. 266, 17954-17958

Gielen et al. (1984) EMBO J 3, 835-845

Gill et al. (1992) Ann. Rev. Entomol. 37: 615-636

Gordon-Kamm et al. (1990) The Plant Cell 2, 603-618

Gould et al. (1991) Plant Physiol. 95, 426-434

Haider et al. (1986) Europ J Biochem 156:531-540

Harlow and Lane (1988) Antibodies: A Manual Laboratory, Cold SpringHarbor Lab Press NY

Harpster et al.(1988), Molecular and General Genetics 212, 182-190

Henikoff and Henikoff (1992) Proc. Natl. Academy Science 89 (10):915-919

Hesse et al. (1989), EMBO J. 8 2453-2461.

Ho et al.(1989). Gene 77, 51-59.

Hofmann et al. (1988) PNAS 85:7844-7848

Höfte et al. (1988) Appl. and Environm. Microbiol. 54, 2010-2017

Hull and Howell (1987) Virology 86, 482-493

Ikemura, 1993, In “Plant Molecular Biology Labfax”, Croy, ed., BiosScientific Publishers Ltd.

Itakura et al. (1977). Science 198, 1056-1063.

Keil et al. (1986), Nucl. Acids Res. 14, 5641-5650.

Klösgen et al. (1989), Mol. Gen. Genet. 217, 155-161.

Klösgen and Weil (1991), Mol. Gen. Genet. 225, 297-304.

Kota et al. (1999) Proc. Natl. Acad. Sci. USA 96, 1840-1845.

Last et al. (1990) Theor. Appl. Genet. 81, 581-588.

Mahillon et al. (1989), FEMS Microbiol. Letters 60, 205-210.

Maxam and Gilbert (1980) Methods in Enzymol. 65, 499-560.

McBride et al. (1995) Bio/Technology 13, 362

McPherson et al. (2000) PCR—Basics: From Background to Bench, FirstEdition, Springer Verlag, Germany

Morris et al. (1999), Biochem. Biophys. Res. Commun. 255, 328-333.

Murray et al. (1989) Nucleic Acids Research 17(2), 477-498.

Nakamura et al. (2000) Nucl. Acids Res. 28, 292.

Needleman and Wunsch algorithm (1970) J. Mol. Biol. 48: 443-453

Neuhaus & Rogers (1998) Plant Mol. Biol. 38, 127-144.

Nielsen, H. J. Engelbrecht, S. Brunak, and G. von Heijne (1996) A neuralnetwork method for identification of prokaryotic and eukaryotic signalpeptides and prediction of their cleavage sites

Odell et al. (1985) Nature 313, 810-812.

Oelmuller et al. (1993) Mol. Gen. Genet. 237, 261-272.

Park et al. (1997) J. Biol. Chem. 272, 6876-6881.

R. D. D. Croy (1993) Plant Molecular Biology Labfax jointly published byBIOS Scientific Publications Ltd (UK) and Blackwell ScientificPublications, UK.

Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, NY

Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual,Third Edition, Cold Spring Harbor Laboratory Press, NY

Sanger et al. (1977) Proc. Natl. Acad. Sci. U S A. 74(12), 5463-5467.

Schnepf and Whiteley (1981) Proc. Natl. Acad. Sci. USA 87: 2893-2897

Selvapandiyan et al. (2001) Appl. and Environm. Microbial 67 (12),5855-5858

Shcherban et al. (1995), Proc. Natl. Acad. Sci USA 92, 9245-9249.

Shimamoto et al (1989) Nature 338, 274-276

Smith and Waterman (1981) Advances in Applied Mathematics 2: 482-489

Stanssens et al.(1989) Nucleic Acids Research 12, 4441-4454.

Sutliff et al. (1991) Plant Malec. Biol. 16, 579-591.

Tavladoraki et al. (1998), FEBS Lett. 426, 62-66.

Terashima et al. (1999), Appl. Microbial. Biotechnol. 52, 516-523.

Vaeck et al. (1987) Nature 328, 33-37.

Van Den Broeck et al. (1985) Nature 313, 358.

Van Rie et al. (1990) Science 247, 72.

Vanderzand (1962) J. Econ. Entomol. 55, 140

Velten et al. (1984) EMBO J 3, 2723-2730

Velten and Schell (1985), Nucleic Acids Research 13, 6981-6998

Verdaguer et al. (1998), Plant Mol. Biol. 37, 1055-1067

Visser et al. (1993) “Domain-Structure Studies of Bacillus thuringiensisCrystal Proteins: A Genetic Approach”, In Bacillus thuringiensis, AnEnvironmental Biopesticide: Theory and Practice, pp.71-88, eds.Entwistle, P. F., Cory, J. S., Bailey, M. J. and Higgs, S., John Wileyand Sons, New York.

Von Heijne, Gunnar (1986) Secretory signal peptide identificationalgorithm described by Gunnar von Heijne Nucleic Acids Research. 14:11,4683-4690

Wada et al. (1990). Nucl. Acids Res. 18, 2367-1411.

Warren (1997) “Advances in Insect Control: The role of transgenicplants”, 1997, editors Carozzi and Koziel, p 109-121, Taylor and FrancisLondon, UK

Waterfield et al. (2001) Trends Microbial 9, 185-91.

White et al. (1989). Trends in Genet. 5, 185-189

Wilbur and Lipmann (1983) Proc. Nat. Acad. Sci. USA 80: 726

Wong et al. (1992), Plant Molec. Biol. 20, 81-93.

Yannisch-Perron et al. (1985) Gene 33, 103-119

Yu et al. (1997) Applied and Environmental Microbioloby 532-536

Zhang et al. (1991) The Plant Cell 3, 1155-1165.

1.-19. (canceled)
 20. An isolated insecticidal protein comprising anamino acid sequence of SEQ ID NO: 4, or an amino acid sequence of thesmallest toxic fragment thereof, wherein said smallest toxic fragment isthe smallest portion of the protein of SEQ ID NO: 4 retaininginsecticidal activity which can be obtained by incubating insectgut-juice extracts with solutions comprising the protein of SEQ ID NO:4,said gut-juice extracts obtained from any one of the following insects:Helicoverpa zea, Heliothis virescens, Ostrinia nubilalis, Spodopterafrugiperda, Agrotis ipsilon, Pectinophora gossypiella, Scirphophagaincertulas, Cnaphalocrocis medinalis, Sesamia inferens, Chilo partellusand Anticarsia gemmatalis.
 21. The protein of claim 20, comprising theamino acid sequence of SEQ ID NO: 4 from amino acid position 55 to aminoacid position 788, from amino acid position 55 to amino acid position455, or from amino acid position 200 to amino acid position
 455. 22. Theprotein of claim 21 comprising the amino acid sequence of SEQ ID NO: 4.23. The protein of any one of claim 22, wherein an Asp or Ala amino acidis inserted as a new second amino acid so that the protein starts with aMet-Asp or Met-Ala dipeptide.
 24. A hybrid or chimeric proteincomprising a toxic fragment of the protein of SEQ ID NO: 4, wherein saidtoxic fragment comprises the amino acid sequence of SEQ ID NO: 4 fromamino acid position 55 to amino acid position 788 or from amino acidposition 200 to amino acid position 455, or wherein said toxic fragmentis the smallest portion of the protein of SEQ ID NO: 4 retaininginsecticidal activity which can be obtained by incubating insectgut-juice extracts with solutions comprising the protein of SEQ ID NO:4,said gut-juice extracts obtained from any one of the following insects:Helicoverpa zea, Heliothis virescens, Ostrinia nubilalis, Spodopterafrugiperda, Agrotis ipsilon, Pectinophora gossypiella, Scirphophagaincertulas, Cnaphalocrocis medinalis, Sesamia inferens, Chilo partellusand Anticarsia gemmatalis.
 25. A method of protecting a plant againstlepidopteran insect damage comprising contacting said plant with theinsecticidal protein of claim
 20. 26. The method of claim 25, whereinsaid protein is applied externally to said plant.
 27. The method of anyone of claim 25, wherein said plant is a maize, cotton, soybean or riceplant.
 28. An insecticidal composition comprising the protein of any oneof claim
 20. 29. The composition of claim 28 which comprises suitablecarriers, diluents, emulsifiers and/or dispersants, which composition,when applied externally to a plant increases resistance to lepidopteraninsect damage compared to control plants to which no such composition isapplied.
 30. A process to control a Lepidopteran cotton, maize, rice orsoybean insect pest, comprising the step of applying a compositioncomprising the protein of claim
 20. 31. A process to control insectsselected from the group consisting of: Scirphophaga spp., Cnaphalocrocisspp., Sesamia spp, Chilo spp., Anticarsia spp., Pseudoplusia spp.,Epinotia spp., Rachiplusia spp, Scirphophaga incertulas, Cnaphalocrocismedinalis, Sesamia inferens, Chilo partellus, Anticarsia gemmatalis,Pseudoplusia includens, Epinotia aporema, Rachiplusia nu, Spodopteraornithogalli, and Plathypena scabra, comprising using an insecticidalprotein comprising the amino acid sequence of SEQ ID NO: 4 or aninsecticidal variant thereof against said insects, wherein said variantis an insecticidal protein selected from the group consisting of: a) aninsecticidal protein with at least 95% sequence identity to the proteinof SEQ ID NO: 4, b) an insecticidal protein comprising the amino acidsequence of SEQ ID NO: 4 from amino acid position 55 to amino acidposition 788 or from amino acid position 200 to amino acid position 455,c) a variant of the protein of SEQ ID NO: 4 or the protein in b) whereinless than 5, or 5-10, amino acids are added, replaced or deleted withoutchanging the insecticidal activity of the protein.
 32. The process ofclaim 31, wherein said insect is selected from the group consisting of:Scirphophaga spp., Cnaphalocrocis spp., Sesamia spp, Chilo spp.,Anticarsia spp., Scirphophaga incertulas, Cnaphalocrocis medinalis,Sesamia inferens, Chilo partellus, and Anticarsia gemmatalis.
 33. Theprocess of claim 31, wherein said insect is selected from the groupconsisting of: Scirphophaga incertulas, Cnaphalocrocis medinalis,Sesamia inferens, Chilo partellus, and Anticarsia gemmatalis.
 34. Aprocess wherein two insecticidal proteins are used to control insectspests, wherein : a) the first protein is an insecticidal proteincomprising the amino acid sequence of SEQ ID NO: 4 or an insecticidalvariant thereof, wherein said variant is an insecticidal proteinselected from the group consisting of: a) an insecticidal protein withat least 95% sequence identity to the protein of SEQ ID NO: 4, b) aninsecticidal protein comprising the amino acid sequence of SEQ ID NO: 4from amino acid position 55 to amino acid position 788 or from aminoacid position 200 to amino acid position 455, c) a variant of theprotein of SEQ ID NO: 4 or the protein in b) wherein less than 5, or5-10, amino acids were added, replaced or deleted without changing theinsecticidal activity of the protein, and b) the second insecticidalprotein is a Cry1F protein, a hybrid derived from a Cry1F protein, aCry1A-type protein, a Cry1Ac protein, a hybrid derived from a Cry1Acprotein, a Cry1Ab protein or an insecticidal fragment thereof, a Cry2Aeprotein, a Cry2Af protein, a Cry2Ag protein, or a VIP3Aa protein. 35.The process of claim 34, wherein said first and second protein areexpressed in maize, rice, cotton or soybean plants, and wherein saidsecond insecticidal protein is: a Cry1Ac protein, a Cry1Ab protein, aCry2Ae protein, or a VIP3Aa protein.
 36. A method for controllingLepidopteran soybean insect pests selected from the group consisting of:Velvet Bean Caterpillar (Anticarsia gemmatalis), Soybean Looper(Pseudoplusia includens), Yellowstriped Armyworm (Spodopteraornithogalli), Pod Borer (Epinotia aporema) and Rachiplusia nu, whichmethod comprises applying to an area or plant to be protected, aninsecticidal protein comprising the amino acid sequence of SEQ ID NO: 4or an insecticidal variant thereof, wherein said variant is aninsecticidal protein selected from the group consisting of: a) aninsecticidal protein with at least 95% sequence identity to the proteinof SEQ ID NO: 4, b) an insecticidal protein comprising the amino acidsequence of SEQ ID NO: 4 from amino acid position 55 to amino acidposition 788 or from amino acid position 200 to amino acid position 455,c) a variant of the protein of SEQ ID NO: 4 or the protein in b) whereinless than 5, or 5-10, amino acids were added, replaced or deletedwithout changing the insecticidal activity of the protein.
 37. Themethod of claim 36 wherein a soybean plant is contacted with saidinsecticidal protein.
 38. The method of claim 36 which comprisesplanting a soybean plant transformed with a gene encoding saidinsecticidal protein, or spraying a composition containing saidinsecticidal protein.